Nucleic acids encoding, and methods of producing, antibodies specific for transforming growth factor (TGF)-β

ABSTRACT

The present disclosure relates, in general, to materials and methods for antibodies specific for transforming growth factor beta (TGFβ), including TGFβ1, TGFβ2 and TGFβ3, and uses of these antibodies in the treatment of subjects having cancer, an eye disease, condition or disorder, fibrosis, including ophthalmic fibrosis or fibrosis of the eye, and other conditions or disorders related to TGFβ expression.

CROSS REFERENCE TO RELATED APPLICATIONS

This is application is a continuation of U.S. patent application Ser.No. 14/808,666, filed Jul. 24, 2015, now U.S. Pat. No. 9,714,285, issuedJul. 25, 2017, which is a continuation of U.S. patent application Ser.No. 14/038,436, filed Sep. 26, 2013, now U.S. Pat. No. 9,145,458, issuedSep. 29, 2015, which is a continuation of U.S. patent application Ser.No. 13/486,983, filed Jun. 1, 2012, now U.S. Pat. No. 8,569,462, issuedOct. 29, 2013, which claims the priority benefit of U.S. ProvisionalPatent Application No. 61/493,230, filed Jun. 3, 2011, hereinincorporated by reference.

FIELD OF THE INVENTION

The present disclosure relates, in general, to materials and methods forantibodies specific for transforming growth factor beta (TGFβ),including TGFβ1, TGFβ2 and/or TGFβ3, and uses of these antibodies in thetreatment of subjects having cancer, an eye disease, condition ordisorder, fibrosis, including fibrosis of the eye or ophthalmicfibroses, and other conditions or disorders related to TGFβ expression.

BACKGROUND

The transforming growth factor beta (TGFβ) protein family consists ofthree distinct isoforms found in mammals (TGFβ1, TGFβ2, and TGFβ3). TheTGFβ proteins activate and regulate multiple gene responses thatinfluence disease states, including cell proliferative, inflammatory,and cardiovascular conditions. TGFβ is a multifunctional cytokineoriginally named for its ability to transform normal fibroblasts tocells capable of anchorage-independent growth. The TGFβ molecules areproduced primarily by hematopoietic and tumor cells and can regulate,i.e., stimulate or inhibit, the growth and differentiation of cells froma variety of both normal and neoplastic tissue origins (Sporn et al.,Science, 233: 532 (1986)), and stimulate the formation and expansion ofvarious stromal cells.

The TGFβs are known to be involved in many proliferative andnon-proliferative cellular processes such as cell proliferation anddifferentiation, embryonic development, extracellular matrix formation,bone development, wound healing, hematopoiesis, and immune andinflammatory responses. See e.g., Pircher et al, Biochem. Biophys. Res.Commun., 136: 30-37 (1986); Wakefield et al., Growth Factors, 1: 203-218(1989); Roberts and Sporn, pp 419-472 in Handbook of ExperimentalPharmacology eds M. B. Sporn & A. B. Roberts (Springer, Heidelberg,1990); Massague et al., Annual Rev. Cell Biol., 6: 597-646 (1990);Singer and Clark, New Eng. J. Med., 341: 738-745 (1999). Also, TGFβ isused in the treatment and prevention of diseases of the intestinalmucosa (WO 2001/24813). TGFβ is also known to have strongimmunosuppressuve effects on various immunologic cell types, includingcytotoxic T lymphocyte (CTL) inhibition (Ranges et al., J. Exp. Med.,166: 991, 1987), Espevik et al., J. Immunol., 140: 2312, 1988),depressed B cell lymphopoiesis and kappa light-chain expression (Lee etal., J. Exp. Med., 166: 1290, 1987), negative regulation ofhematopoiesis (Sing et al., Blood, 72: 1504, 1988), down-regulation ofHLA-DR expression on tumor cells (Czarniecki et al., J. Immunol., 140:4217, 1988), and inhibition of the proliferation of antigen-activated Blymphocytes in response to B-cell growth factor (Petit-Koskas et al.,Eur. J. Immunol., 18: 111, 1988). See also U.S. Pat. No. 7,527,791.

Antibodies to TGFβ have been described in U.S. Pat. Nos. 7,527,791;7,927,593; 7,494,651; 7,369,111; 7,151,169; 6,492,497; 6,419,928;6,090,383; 5,783,185; 5,772,998; 5,571,714; and 7,723,486.

SUMMARY OF THE INVENTION

The present disclosure provides methods and compositions for thetreatment of disease or disorders associated with TGFβ expression. Thedisclosure provides antibodies that bind TGFβ1, TGFβ2 and TGFβ3. It isprovided that the antibodies described herein can have differentialaffinity for any or all of the TGFβ isoforms. Further, it was discoveredherein that the disclosed TGFβ-specific antibodies unexpectedly modulateimmune cells in tumors (e.g., infiltrate into tumors) and arecontemplated for treatment of tumors associated with TGFβ expression, aswell as other conditions or disorders associated with TGFβ expression.

In one aspect, the disclosure provides an antibody that bindstransforming growth factor beta (TGFβ)1, TGFβ2 and TGFβ3 comprising: (a)a heavy chain complementary determining repeat (CDR)1 amino acidsequence set forth in Table 1 or SEQ ID NOs: 13, 19 and 25, or a variantthereof in which one or two amino acids have been changed; (b) a heavychain CDR2 amino acid sequence set forth in Table 1 or SEQ ID NOs: 14,20 and 26 that is from the same heavy chain variable region as (a), or avariant thereof in which one or two amino acids have been changed; and(c) a heavy chain CDR3 amino acid sequence set forth in Table 1 or SEQID NOs: 15, 21 and 27 that is from the same heavy chain variable regionas (a), or a variant thereof in which one or two amino acids have beenchanged.

In a related aspect, the disclosure provides an antibody that bindstransforming growth factor beta (TGFβ)1, TGFβ2 and TGFβ3 comprising: (a)a heavy chain CDR1 amino acid sequence set forth in Table 1 or SEQ IDNOs: 13, 19 and 25, or a variant thereof having at least 70% identitythereto; (b) a heavy chain CDR2 amino acid sequence set forth in Table 1or SEQ ID NOs: 14, 20 and 26 that is from the same heavy chain variableregion as (a), or a variant thereof having at least 70% identitythereto; and (c) a heavy chain CDR3 amino acid sequence set forth inTable 1 or SEQ ID NOs: 15, 21 and 27 that is from the same heavy chainvariable region as (a), or a variant thereof having at least 70%identity thereto.

In a further aspect, the disclosure contemplates an antibody that bindstransforming growth factor beta (TGFβ)1, TGFβ2 and TGFβ3 comprising: (a)a heavy chain CDR1 amino acid sequence set forth in Table 1 or SEQ IDNOs: 13, 19 and 25, or a variant thereof having at least 70% identitythereto; (b) an independently selected heavy chain CDR2 amino acidsequence set forth in Table 1 or SEQ ID NOs: 14, 20 and 26, or a variantthereof having at least 70% identity thereto; and (c) an independentlyselected heavy chain CDR3 amino acid sequence set forth in Table 1 orSEQ ID NOs: 15, 21 and 27, or a variant thereof having at least 70%identity thereto.

In certain embodiments, at least two of the heavy chain CDR1, CDR2 orCDR3 amino acid sequences are set forth in Table 1 or SEQ ID NOs: 13-15,19-21 and 25-27. In a related embodiment, three of the heavy chain CDR1,CDR2 and CDR3 amino acid sequences are set forth in Table 1 or SEQ IDNOs: 13-15, 19-21 and 25-27.

In some embodiments, it is contemplated that the antibody comprises anamino acid sequence at least 85% identical to a heavy chain variableregion amino acid sequence set forth in Table 1 or SEQ ID NOs: 2, 6 and10. In a related embodiment, the antibody comprises an amino acidsequence at least 95% identical to a heavy chain variable region aminoacid sequence set forth in Table 1 or SEQ ID NOs: 2, 6 and 10.

In still other embodiments, the antibody comprises a polypeptidesequence having an amino acid sequence at least 70% identical over allthree HCDRs in a heavy chain variable region, the amino acid sequencesof HCDR1, HCDR2 and HCDR3 set forth in SEQ ID NOs: 13-15, 19-21 and25-27.

In certain embodiments, one or more heavy chain framework amino acidshave been replaced with corresponding amino acid(s) from another humanantibody amino acid sequence.

It is contemplated that an antibody described herein further comprisesany one of the light chain CDR amino acid sequences set forth in Table 1or SEQ ID NOs: 16-18, 22-24 and 28-30. In some embodiments, an antibodycomprises at least two of the light chain CDR amino acid sequences setforth in Table 1 or SEQ ID NOs: 16-18, 22-24 and 28-30. In otherembodiments, an antibody comprises at least three of the light chain CDRamino acid sequences set forth in Table 1 or SEQ ID NOs: 16-18, 22-24and 28-30.

In another aspect, an antibody described herein comprises (a) a lightchain CDR1 amino acid sequence set forth in Table 1 or SEQ ID NOs: 16,22 and 28, or a variant thereof in which one or two amino acids havebeen changed; (b) a light chain CDR2 amino acid sequence set forth inTable 1 or SEQ ID NOs: 17, 23 and 29 that is from the same light chainvariable region as (a), or a variant thereof in which one or two aminoacids have been changed; and (c) a light chain CDR3 amino acid sequenceset forth in Table 1 or SEQ ID NOs: 18, 24 and 30 that is from the samelight chain variable region as (a), or a variant thereof in which one ortwo amino acids have been changed.

In alternative embodiments, an antibody contemplated herein comprises:(a) a light chain CDR1 amino acid sequence set forth in Table 1 or SEQID NOs: 16, 22 and 28, or a variant thereof in which one or two aminoacids have been changed; (b) an independently selected light chain CDR2amino acid sequence set forth in Table 1 or SEQ ID NOs: 17, 23 and 29,or a variant thereof in which one or two amino acids have been changed;and (c) an independently selected light chain CDR3 amino acid sequenceset forth in Table 1 or SEQ ID NOs: 18, 24 and 30, or a variant thereofin which one or two amino acids have been changed.

In certain embodiments, at least two of the light chain CDR1, CDR2 orCDR3 amino acid sequences are set forth in Table 1 or SEQ ID NOs: 16-18,22-24 and 28-30.

It is further contemplated that an antibody described herein comprises apolypeptide sequence having an amino acid sequence at least 70%identical over all three LCDRs of a light chain variable region, theamino acid sequences of LCDR1, LCDR2 and LCDR3 set forth in SEQ ID NOs:16-18, 22-24 and 28-30.

In one embodiment, an antibody contemplated herein comprises an aminoacid sequence at least 70% identical to a light chain variable regionamino acid sequence set forth in Table 1 or SEQ ID NOs: 4, 8 and 12. Ina related embodiment, the antibody comprises an amino acid sequence atleast 85% identical to a light chain variable region amino acid sequenceset forth in Table 1 or SEQ ID NOs: 4, 8 and 12. In a furtherembodiment, the antibody comprises an amino acid sequence at least 95%identical to a light chain variable region amino acid sequence set forthin Table 1 or SEQ ID NOs: 4, 8 and 12. In still another embodiment, theantibody comprises a light chain variable region amino acid sequence setforth in Table 1 or SEQ ID NOs: 4, 8 and 12.

In a further embodiment, an antibody described herein comprises (i) anamino acid sequence at least 70% identical over all three LCDRs, of alight chain variable region, the amino acid sequences of LCDR1, LCDR2and LCDR3 set forth in SEQ ID NOs: 16-18, 22-24 and 28-30 and (ii) anamino acid sequence at least 70% identical over all three HCDRs of aheavy chain variable region, the amino acid sequences of HCDR1, HCDR2and HCDR3 set forth in SEQ ID NOs: 13-15, 19-21 and 25-27.

In another aspect, the disclosure provides an antibody that bindstransforming growth factor beta (TGFβ)1, TGFβ2 and TGFβ3 comprising alight chain variable region and/or a heavy chain variable region,wherein (a) the light chain variable region comprises at least a CDR1selected from SEQ ID NOs: 16, 22 and 28 or sequences at least 80%identical thereto, a CDR2 selected from SEQ ID NOs: 17, 23 and 29 orsequences at least 80% identical thereto, and/or a CDR3 selected fromSEQ ID NOs: 18, 24 and 30 or sequences at least 80% identical thereto;and/or wherein (b) the heavy chain variable region comprises at least aCDR1 selected from SEQ ID NOs: 13, 19 and 25 or sequences at least 80%identical thereto, a CDR2 selected from SEQ ID NOs: 14, 20 and 26 orsequences at least 80% identical thereto, and/or a CDR3 selected fromSEQ ID NOs: 15, 21 and 27 or sequences at least 80% identical thereto.In one embodiment, the light chain variable region comprises at least aCDR1 selected from SEQ ID NO: 16 or sequences at least 90% identicalthereto, a CDR2 selected from SEQ ID NO: 17 or sequences at least 90%identical thereto, and a CDR3 selected from SEQ ID NO: 18 or sequencesat least 90% identical thereto; and/or the heavy chain variable regioncomprises at least a CDR1 selected from SEQ ID NO: 13 or sequences atleast 90% identical thereto, a CDR2 selected from SEQ ID NO: 14 orsequences at least 90% identical thereto, and a CDR3 selected from SEQID NO: 15 or sequences at least 90% identical thereto.

In a related embodiment, the light chain variable region comprises atleast a CDR1 selected from SEQ ID NO: 22 or sequences at least 90%identical thereto, a CDR2 selected from SEQ ID NO: 23 or sequences atleast 90% identical thereto, and a CDR3 selected from SEQ ID NO: 24 orsequences at least 90% identical thereto; and/or the heavy chainvariable region comprises at least a CDR1 selected from SEQ ID NO: 19 orsequences at least 90% identical thereto, a CDR2 selected from SEQ IDNO: 20 or sequences at least 90% identical thereto, and a CDR3 selectedfrom SEQ ID NO: 21 or sequences at least 90% identical thereto.

In certain embodiments, the light chain variable region comprises atleast a CDR1 selected from SEQ ID NO: 28 or sequences at least 90%identical thereto, a CDR2 selected from SEQ ID NO: 29 or sequences atleast 90% identical thereto, and a CDR3 selected from SEQ ID NO: 30 orsequences at least 90% identical thereto; and/or the heavy chainvariable region comprises at least a CDR1 selected from SEQ ID NO: 25 orsequences at least 90% identical thereto, a CDR2 selected from SEQ IDNO: 26 or sequences at least 90% identical thereto, and a CDR3 selectedfrom SEQ ID NO: 27 or sequences at least 90% identical thereto.

It is contemplated that the percent identity of any one of the aboveantibody sequences can be at least 70%, 75%, 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96% , 97%, 98%,99% or more identical to a heavy or light chain variable region or anyof HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 or LCDR3 disclosed herein.

In some embodiments, an antibody of the disclosure further comprises aheavy chain constant region, wherein the heavy chain constant region isa modified or unmodified IgG, IgM, IgA, IgD, IgE, a fragment thereof, orcombinations thereof.

In certain embodiments, an antibody is provided in which one or morelight chain framework amino acids have been replaced with correspondingamino acid(s) from another human antibody amino acid sequence.

In one aspect, the antibody of the disclosure is selected from the groupconsisting of XPA.42.089, XPA.42.068 and XPA.42.681. Heavy and lightchain amino acid sequences of XPA.42.089 are set out in SEQ ID NOs: 6and 8, respectively. Heavy and light chain amino acid sequences ofXPA.42.068 are set out in SEQ ID NOs: 2 and 4, respectively, and heavyand light chain amino acid sequences of XPA.42.681 are set out in SEQ IDNOs: 10 and 12, respectively.

In one embodiment, an antibody described herein further comprises ahuman light chain constant region attached to said light chain variableregion. In some embodiments, the light chain constant region is amodified or unmodified lambda light chain constant region, a kappa lightchain constant region, a fragment thereof, or combinations thereof.

In a preferred embodiment, the disclosure provides an antibody specificfor TGFβ1, TGFβ2 and TGFβ3 with an affinity Kd of 10⁻⁶ M or less. Inexemplary embodiments, an anti-TGFβ antibody described herein binds atleast one isoform of TGFβ with an affinity of 10⁻⁶ M, 10⁻⁷ M, 10⁻⁸ M,10⁻⁹ M or less, or optionally binds two TGFβ isoforms, or all of TGFβ1,2, or 3 with an affinity of 10⁻⁶ M, 10⁻⁷ M, 10⁻⁸ M, 10⁻⁹ M, 10⁻¹⁰ M,10⁻¹¹ M, or 10⁻¹² M or less for one or more of the isoforms. In otherembodiments, an antibody described herein binds to TGFβ1 and TGFβ2 withat least 2-50 fold, 10-100 fold, 2-fold, 5-fold, 10-fold, 25-fold,50-fold or 100-fold, or 20-50%, 50-100%, 20%, 25%, 30%, 40%, 50%, 60%,70%, 80%, 90% or 100% higher affinity (e.g., preferentially binds toTGFβ1 and TGFβ2) compared to binding to TGFβ3. Alternatively, anantibody described herein, binds each of TGFβ isoforms TGFβ1, TGFβ2 andTGFβ3 with an affinity within 3-fold, 5-fold or 10-fold of each other.In certain embodiments, the antibody binds to TGFβ1 and TGFβ2 withgreater affinity than TGFβ3. In certain embodiments, the affinity ismeasured by surface plasmon resonance or KINEXA assay.

In some embodiments, the antibody neutralizes activity of TGFβ1 andTGFβ2 to a greater extent than TGFβ3. In some embodiments, antibodyneutralization of TGFβ1 and TGFβ2 is at least 2-50 fold, 10-100 fold,2-fold, 5-fold, 10-fold, 25-fold, 50-fold or 100-fold, or 20-50%,50-100%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% more potentthat neutralization of TGFβ3. Exemplary neutralization assayscontemplated herein include, but are not limited to, an interleukin-11release assay and an HT-2 cell proliferation assay. In addtion, a TGFβactivity assay can be carried out to determine if an antibody disclosedherein inhibits one TGFβ isoform preferentially, including a pSMADphosphorylation assay or an rhLAP binding assay. In a furtherembodiment, the antibody has a lower IC50 (i.e., better binding, greaterpotency) for TGFβ1 and TGFβ2 compared to TGFβ3.

In another aspect, the disclosure provides an isolated nucleic acidmolecule comprising a nucleotide sequence that encodes the heavy chainand/or light chain as described herein.

In a further aspect, the disclosure provides an expression vectorcomprising a nucleic acid molecule contemplated herein operably linkedto an expression control sequence. Also contemplated is a host cellcomprising an expression vector or a nucleic acid molecule of thedisclosure. In certain embodiments, the disclosure provides a host cellcomprising a nucleic acid molecule encoding a heavy chain and a lightchain variable region, wherein the heavy chain and light chain nucleicacids are expressed by different nucleic acids or on the same nucleicacid.

In a related aspect, the disclosure provides a method of using the hostcell as described herein to produce an antibody, the method comprisingculturing the host cell under suitable conditions and recovering saidantibody. Also provided is an antibody produced by the method disclosedherein.

The disclosure further contemplates a sterile pharmaceutical compositioncomprising the antibody as disclosed herein and a pharmaceuticallyacceptable carrier.

In another aspect, the disclosure provides a method for treating adisease, condition or disorder associated with TGFβ expressioncomprising the step of administering to a subject in need thereof atherapeutically effective amount of an antibody or a pharmaceuticalcomposition contemplated herein. In certain embodiments, the disease,condition or disorder is selected from the group consisting of a cancer,an eye (e.g., ocular, optic, ophthalmic or ophthalmological) disease,condition or disorder, a disease condition or disorder associated withfibrosis, e.g., fibroproliferative diseases, conditions or disorders, ordisease, conditions or disorders having an associated fibrosis.

Fibroproliferative diseases, conditions or disorders or diseases havingan associated fibrosis include those that affect any organ or tissue inthe body, including, but not limited to the skin, lung, kidney, heart,brain and eye. Fibroproliferative diseases, conditions or disorders ordiseases having an associated fibrosis include, but are not limited topulmonary fibrosis, idiopathic pulmonary fibrosis, peribronchiolarfibrosis, interstitial lung disease, chronic obstructive pulmonarydisease (COPD), small airway disease (e.g., obstructive bronchiolitis),emphysema, adult or acute respiratory distress syndrome (ARDS), acutelung injury (ALI), pulmonary fibrosis due to infectious or toxic agents,kidney fibrosis, glomerulonephritis (GN) of all etiologies, mesangialproliferative GN, immune GN, crescentic GN, glomerulosclerosis,tubulointerstitial injury, renal interstitial fibrosis, renal fibrosisand all causes of renal interstitial fibrosis, renal fibrosis resultingfrom complications of drug exposure, including cyclosporin treatment oftransplant recipients, HIV-associated nephropathy, transplantnecropathy, diabetic kidney disease, diabetic nephropathy, nephrogenicsystemic fibrosis, diabetes, idiopathic retroperitoneal fibrosis,scleroderma, liver fibrosis, hepatic diseases associated with excessivescarring and progressive sclerosis, liver cirrhosis due to alletiologies, disorders of the biliary tree, hepatic dysfunctionattributable to infections, fibrocystic diseases, cardiovasculardiseases, congestive heart failure, dilated cardiomyopathy, myocarditis,vascular stenosis cardiac fibrosis, post-infarction cardiac fibrosis,post myocardial infarction, left ventricular hypertrophy, veno-occlusivedisease, restenosis, post-angioplasty restenosis, arteriovenous graftfailure, atherosclerosis, hypertension, hypertensive heart disease,cardiac hypertrophy, hypertrophic cardiomyopathy, heart failure, diseaseof the aorta, progressive systemic sclerosis; polymyositis; systemiclupus erythematosus; dermatomyositis, fascists, Raynaud's syndrome,rheumatoid arthritis, proliferative vitreoretinopathy, vitreoretinopathyof any etiology, fibrosis associated with ocular surgery, treatment ofglaucoma, retinal reattachment, cataract extraction, or drainageprocedures of any kind, scarring in the cornea and conjunctiva, fibrosisin the corneal endothelium, alkali burn, (e.g., alkali burn to thecornea) post-cataract surgery fibrosis of the lens capsule, excessscarring in the tissue around the extraocular muscles in the strabismussurgery, anterior subcapsular cataract and posterior capsuleopacification, anterior segment fibrotic diseases of the eye, fibrosisof the corneal stroma, fibrosis associated with corneal opacification,fibrosis of the trabecular network, fibrosis associated with glaucoma,posterior segment fibrotic diseases of the eye, fibrovascular scarring,fibrosis in retinal or choroidal vasculature of the eye, retinalfibrosis, epiretinal fibrosis, retinal gliosis, subretinal fibrosis,fibrosis associated with age related macular degeneration, post-retinaland glaucoma surgery, tractional retinal detachment in association withcontraction of the tissue in diabetic retinopathy, Peyronie's disease,systemic sclerosis, post-spinal cord injury, osteoporosis,Camurati-Engelmann disease, Crohn's disease, scarring, Marfan syndrome,premature ovarian failure, Alzheimer's Disease, Parkinson's Disease,fibrosis due to surgical incisions or mechanical trauma, fibrosisassociated with ocular surgery, and excessive or hypertrophic scar orkeloid formation in the dermis occurring during wound healing resultingfrom trauma or surgical wounds.

Exemplary eye diseases (e.g., ocular, optic, ophthalmic orophthalmological diseases), conditions or disorders, include but are notlimited to, fibroproliferative disorders, fibrosis of the eye,ophthalmic fibroses, retinal dysfunction, fibrosis associated withretinal dysfunction, wet or dry macular degeneration, proliferativevitreoretinopathy, vitreoretinopathy of any etiology, fibrosisassociated with ocular surgery such as treatment of glaucoma, retinalreattachment, cataract extraction, or drainage procedures of any kind,scarring in the cornea and conjunctiva, fibrosis in the cornealendothelium, alkali burn (e.g., alkali burn to the cornea),post-cataract surgery fibrosis of the lens capsule, excess scarring inthe tissue around the extraocular muscles in the strabismus surgery,anterior subcapsular cataract and posterior capsule opacification,anterior segment fibrotic diseases of the eye, fibrosis of the cornealstroma (e.g., associated with corneal opacification), fibrosis of thetrabecular network (e.g., associated with glaucoma), posterior segmentfibrotic diseases of the eye, fibrovascular scarring (e.g., in retinalor choroidal vasculature of the eye), retinal fibrosis, epiretinalfibrosis, retinal gliosis, subretinal fibrosis (e.g., associated withage related macular degeneration), fibrosis associated with post-retinaland glaucoma surgery, tractional retinal detachment in association withcontraction of the tissue in diabetic retinopathy.

Exemplary fibroproliferative disease, condition, or disorders of theeye, fibrosis of the eye, ocular fibrosis or ophthalmic fibrosesinclude, but are not limited to, proliferative vitreoretinopathy,vitreoretinopathy of any etiology, fibrosis associated with retinaldysfunction, fibrosis associated with wet or dry macular degeneration,fibrosis associated with ocular surgery such as treatment of glaucoma,retinal reattachment, cataract extraction, or drainage procedures of anykind, scarring in the cornea and conjunctiva, fibrosis in the cornealendothelium, fibrosis associated with alkali burn, post-cataract surgeryfibrosis of the lens capsule, excess scarring the tissue around theextraocular muscles in the strabismus surgery, anterior subcapsularcataract and posterior capsule opacification, anterior segment fibroticdiseases of the eye, fibrosis of the corneal stroma (e.g., associatedwith corneal opacification), fibrosis of the trabecular network (e.g.,associated with glaucoma), posterior segment fibrotic diseases of theeye, fibrovascular scarring (e.g., in retinal or choroidal vasculatureof the eye), retinal fibrosis, epiretinal fibrosis, retinal gliosis,subretinal fibrosis (e.g., associated with age related maculardegeneration), fibrosis associated with post-retinal and glaucomasurgery, tractional retinal detachment in association with contractionof the tissue in diabetic retinopathy.

In various embodiments, the fibroproliferative disease, condition, ordisorders of the eye is selected from the group consisting ofproliferative vitreoretinopathy, fibrosis associated with ocularsurgery, post-cataract surgery fibrosis of the lens, fibrosis of thecorneal stroma and alkali burn.

In a related aspect, the disclosure provides a method for treatingcancer comprising administering to a subject in need thereof atherapeutically effective amount of an antibody or a pharmaceuticalcomposition contemplated herein. In certain embodiments, the cancer isselected from the group consisting of lung cancer, prostate cancer,breast cancer, hepatocellular cancer, esophageal cancer, colorectalcancer, pancreatic cancer, bladder cancer, kidney cancer, ovariancancer, stomach cancer, fibrotic cancer, glioma and melanoma.

In some embodiments, the antibody or composition increases the number ofnatural killer (NK) cells in a tumor. In various embodiments, theantibody or composition increases cytolytic activity of NK cells. Forexample, in various embodiments, the antibody or composition describedherein increases perforin and granzyme production by NK cells. In oneembodiment, the antibody is XPA.42.089 or XPA.42.681.

In various embodiments, the antibody or composition described hereindecreases the number of regulatory T cells in a tumor and/or inhibitsregulatory T cell function. For example, in various embodiments, theantibody or composition described herein inhibits inhibits the abilityof Tregs to down-regulate an immune response or to migrate to a site ofan immune response.

In various embodiments, the antibody or composition increases the numberof cytotoxic T cells in a tumor and/or enhances CTL activity, e.g.,boosts, increases or promotes CTL activity. For example, in variousembodiments, the antibody or composition described herein increasesperforin and granzyme production by CTL and increases cytolytic activityof the CTL. In one embodiment, the antibody is XPA.42.068, XPA.42.089 orXPA.42.681.

In another embodiment, the antibody or composition decreases the numberof monocyte-derived stem cells (MDSC) in a tumor and/or inhibits MDSCfunction. For example, in various embodiments, the antibody orcomposition described herein inhibits the ability of MDSCs to suppressan immune response, inhibits immune suppressive activity of MDSCs,and/or inhibits the ability of MDSCs to promote expansion and/orfunction of Tregs. In various embodiments, the antibody is selected fromthe group consisting of XPA.42.089, XPA.42.068 and XPA.42.681.

In various embodiments, the antibody decreases the number of dendriticcells (DC) in a tumor and/or inhibits the tolerogenic function (e.g.,tolerogenic effect) of dendritic cells. For example, in variousembodiments, the antibody or composition described herein decreases thetoleragenic effect of CD8+ dendritic cells. In one embodiment, theantibody is XPA.42.089 or XPA.42.681.

In another aspect, the disclosure provides a method for treatingfibrosis comprising administering to a subject in need thereof atherapeutically effective amount of an antibody or a pharmaceuticalcomposition contemplated herein.

In various embodiments, the antibody is administered with a secondagent. In one embodiment, the second agent is selected from the groupconsisting of an extracellular matrix degrading protein, ananti-fibrotic agent, surgical therapy, chemotherapy, a cytotoxic agent,or radiation therapy. Exemplary second agents are disclosed in greaterdetail in the Detailed Description.

In various embodiments, therapy is administered on a period basis, forexample, hourly, daily, weekly, every 2 weeks, every 3 weeks, monthly,or at a longer interval. In a related embodiment, in exemplarytreatments, the antibody disclosed herein may be administered at a doseof about 1 mg/day, 5 mg/day, 10 mg/day, 20 mg/day, 50 mg/day, 75 mg/day,100 mg/day, 150 mg/day, 200 mg/day, 250 mg/day, 500 mg/day or 1000mg/day. These concentrations may be administered as a single dosage formor as multiple doses.

Also contemplated is a composition comprising any of the foregoingantibodies or compositions of the disclosure that bind TGFβ, or usethereof in preparation of a medicament, for treatment of any of thedisorders described herein associated with TGFβ expression. Syringes,e.g., single use or pre-filled syringes, sterile sealed containers, e.g.vials, bottle, vessel, and/or kits or packages comprising any of theforegoing antibodies or compositions, optionally with suitableinstructions for use, are also contemplated.

It is understood that each feature or embodiment, or combination,described herein is a non-limiting, illustrative example of any of theaspects of the invention and, as such, is meant to be combinable withany other feature or embodiment, or combination, described herein. Forexample, where features are described with language such as “oneembodiment”, “some embodiments”, “certain embodiments”, “furtherembodiment”, “specific exemplary embodiments”, and/or “anotherembodiment”, each of these types of embodiments is a non-limitingexample of a feature that is intended to be combined with any otherfeature, or combination of features, described herein without having tolist every possible combination. Such features or combinations offeatures apply to any of the aspects of the invention. Where examples ofvalues falling within ranges are disclosed, any of these examples arecontemplated as possible endpoints of a range, any and all numericvalues between such endpoints are contemplated, and any and allcombinations of upper and lower endpoints are envisioned.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing competition of TGFβ1 binding to rhLAP by TGFβantibodies.

FIGS. 2A-2C show neutralization of pSMAD signaling in cells by TGFβantibodies. (FIG. 2A) TGFβ1; (FIG. 2B) TGFβ2; (FIG. 2C) TGFβ3.

FIG. 3 is a graph showing inhibition of regulatory T cells (Treg) byTGFβ antibodies.

FIG. 4 is a graph showing tumor inhibition in a xenograft mouse model byTGFβ antibodies.

FIG. 5 is a graph showing tumor inhibition in a xenograft mouse model byTGFβ antibodies.

FIG. 6 is a graph showing tumor inhibition in a syngeneic mouse model byTGFβ antibodies.

FIG. 7 is a graph showing tumor inhibition in a syngeneic mouse model byTGFβ antibodies.

FIG. 8 is a graph showing tumor inhibition in a syngeneic mouse model byTGFβ antibodies.

FIG. 9 is a graph showing tumor inhibition in a syngeneic mouse model byTGFβ antibodies.

FIG. 10 is a graph showing the in vivo effect of TGFβ antibodies onnatural killer cells in tumors, in a syngeneic mouse tumor model.

FIG. 11 is a graph showing the in vivo effect of TGFβ antibodies onmyeloid-derived suppressor cells in tumors, in a syngeneic mouse model.

FIG. 12 is a graph showing the in vivo effect of TGFβ antibodies ondendritic cells in tumors in a syngeneic mouse tumor model.

FIG. 13 is a graph showing the in vivo effect of TGFβ antibodies onregulatory T cells in tumors in a syngeneic mouse tumor model.

FIG. 14 is a graph showing the in vivo effect of TGFβ antibodies oncytotoxic T cells in tumors in a syngeneic mouse tumor model.

FIG. 15 is a graph showing the in vitro effects of TGFβ antibodies on NKcell cytolytic activity.

FIG. 16 is a graph showing the effect of TGFβ antibodies on T cellproliferation.

FIGS. 17A-17B show the effect of TGFβ antibodies on CTL activationevaluated by expression of granzyme B (GzmB) (FIG. 17A) and perforin(FIG. 17B).

FIG. 18 is a graph showing the effect of TGFβ antibodies on serum bloodurea nitrogen (BUN) levels in CsA treated or control animalsadministered TGFβ antibodies.

FIG. 19 is a graph showing the effect of TGFβ antibodies on albuminaccumulation, which is characteristic of glomerular dysfunctional in thediseased kidney, in the urine of CsA treated or control animalsadministered TGFβ antibodies.

FIG. 20 is a graph showing the effect of TGFβ antibodies on levels ofurine type IV Collagen, which reflect the extent of ECM deposition andfibrosis in the kidneys, in the urine of CsA treated or control animalsadministered TGFβ antibodies.

FIGS. 21A-B show the effect of TGFβ antibodies on expression of genesinvolved in fibrosis as assessed by Quantitative RT-PCR performed onkidney tissue. Effects on TGF-β1 expression (FIG. 21A) and type IIIcollagen (FIG. 21B) were assessed in CsA treated or control animalsadministered TGFβ antibodies.

FIG. 22 is a graph showing the effect of TGFβ antibodies on increase inpSMAD2 in retinal pigment epithelium (RPE) cells after administration ofTGFβ1.

FIG. 23 is a Table (Table 1) setting out the nucleotide and amino acidsequences, including heavy chain and light chain variable regions andCDR sequences, of the TGFβ-specific antibodies.

DETAILED DESCRIPTION

The present disclosure provides therapeutics to treat conditions ordisorders associated with TGFβ expression, for example, cancer andfibrosis. The present disclosure provides molecules or agents thatinteract with TGFβ and inhibit one or more of its functional effects,such as for example signaling through binding partners of TGFβ. Thecompositions disclosed herein advantageously have the ability tomodulate immune cell activity in tumors, thereby providing, in oneaspect, a method to treat cancer by affecting a cell population thatdirectly or indirectly affects growth of the tumor.

In order that the disclosure may be more completely understood, severaldefinitions are set forth.

As used herein, “target” or “target antigen” refers to any or all of theTGF-β molecules, including TGFβ1, TGFβ2 and TGFβ3.

As used herein “TGFβ” refers to any one or more isoforms of TGFβ,including TGFβ1, TGFβ2 and TGFβ3 or variants thereof. Likewise, the term“TGFβ receptor,” unless otherwise indicated, refers to any receptor thatbinds at least one TGFβ isoform

As used herein, the “desired biological activity” of an anti-targetantibody is the ability to bind to TGFβ and inhibit one or more of itsfunctional effects.

As used herein, a “condition” or “disorder associated with targetexpression” is a condition or disorder in which target activity isdetrimental and includes diseases and other disorders in which highlevels of target have been shown to be or are suspected of being eitherresponsible for the pathophysiology of the disorder or a factor thatcontributes to a worsening of the disorder, as well as diseases andother disorders in which high levels of target expression are associatedwith undesirable clinical signs or symptoms. Such disorders may beevidenced, for example, by an increase in the levels of target secretedand/or on the cell surface and/or increased signaling in the affectedcells or tissues of a subject suffering from the disorder. An increasein target levels may be detected, for example, using an target specificantibody as described herein.

Exemplary diseases, conditions or disorders associated with TGFβexpression that can be treated with an antibody substance that bindsTGFβ (e.g., antibodies of the present disclosure) include cancers, suchas lung cancer, prostate cancer, breast cancer, hepatocellular cancer,esophageal cancer, colorectal cancer, pancreatic cancer, bladder cancer,kidney cancer, ovarian cancer, stomach cancer, fibrotic cancer, glioma,and melanoma, eye (e.g., ocular, optic, ophthalmic or ophthalmological)diseases, conditions or disorders, disease conditions or disordersassociated with fibrosis, e.g., fibroproliferative diseases, conditionsor disorders, or diseases, conditions or disorders having an associatedfibrosis.

Fibroproliferative diseases, conditions or disorders, or diseasesconditions or disorders having an associated fibrosis, include thosethat affect any organ or tissue in the body, including, but not limitedto the skin, lung, kidney, heart, brain and eye. Fibroproliferativediseases, conditions or disorders or diseases having an associatedfibrosis include but are not limited to, pulmonary fibrosis, idiopathicpulmonary fibrosis, peribronchiolar fibrosis, interstitial lung disease,chronic obstructive pulmonary disease (COPD), small airway disease(e.g., obstructive bronchiolitis), emphysema, adult or acute respiratorydistress syndrome (ARDS), acute lung injury (ALI), pulmonary fibrosisdue to infectious or toxic agents, kidney fibrosis, glomerulonephritis(GN) of all etiologies, e.g., mesangial proliferative GN, immune GN, andcrescentic GN, glomerulosclerosis, tubulointerstitial injury, renalinterstitial fibrosis, renal fibrosis and all causes of renalinterstitial fibrosis, renal fibrosis resulting from complications ofdrug exposure, including cyclosporin treatment of transplant recipients,e.g. cyclosporin treatment, HIV-associated nephropathy, transplantnecropathy, diabetic kidney disease (e.g., diabetic nephropathy),nephrogenic systemic fibrosis, diabetes, idiopathic retroperitonealfibrosis, scleroderma, liver fibrosis, hepatic diseases associated withexcessive scarring and progressive sclerosis, including liver cirrhosisdue to all etiologies, disorders of the biliary tree, hepaticdysfunction attributable to infections, fibrocystic diseases,cardiovascular diseases, such as congestive heart failure; dilatedcardiomyopathy, myocarditis, vascular stenosis cardiac fibrosis (e.g.,post-infarction cardiac fibrosis), post myocardial infarction, leftventricular hypertrophy, veno-occlusive disease, restenosis (e.g.,post-angioplasty restenosis), arteriovenous graft failure,atherosclerosis, hypertension, hypertensive heart disease, cardiachypertrophy, hypertrophic cardiomyopathy, heart failure, disease of theaorta, progressive systemic sclerosis, polymyositis, systemic lupuserythematosus, dermatomyositis, fascists, Raynaud's syndrome, rheumatoidarthritis, proliferative vitreoretinopathy, vitreoretinopathy of anyetiology or fibrosis associated with ocular surgery such as treatment ofglaucoma, retinal reattachment, cataract extraction, or drainageprocedures of any kind, scarring in the cornea and conjunctiva, fibrosisin the corneal endothelium, alkali burn (e.g., alkali burn to thecornea), post-cataract surgery fibrosis of the lens capsule, excessscarring the tissue around the extraocular muscles in the strabismussurgery, anterior subcapsular cataract and posterior capsuleopacification, anterior segment fibrotic diseases of the eye, fibrosisof the corneal stroma (e.g., associated with corneal opacification),fibrosis of the trabecular network (e.g., associated with glaucoma),posterior segment fibrotic diseases of the eye, fibrovascular scarring(e.g., in retinal or choroidal vasculature of the eye), retinalfibrosis, epiretinal fibrosis, retinal gliosis, subretinal fibrosis(e.g., associated with age related macular degeneration), fibrosisassociated with post-retinal and glaucoma surgery, tractional retinaldetachment in association with contraction of the tissue in diabeticretinopathy, Peyronie's disease, systemic sclerosis, post-spinal cordinjury, osteoporosis, Camurati-Engelmann disease, Crohn's disease,scarring, Marfan syndrome, premature ovarian failure, Alzheimer'sDisease and Parkinson's Disease, fibrosis due to surgical incisions ormechanical trauma, fibrosis associated with ocular surgery; andexcessive or hypertrophic scar or keloid formation in the dermisoccurring during wound healing resulting from trauma or surgical wounds.

Exemplary eye diseases, (e.g., ocular, optic, ophthalmic orophthalmological diseases), conditions or disorders, include but are notlimited to, fibroproliferative disorders, fibrosis of the eye,ophthalmic fibroses, retinal dysfunction, fibrosis associated withretinal dysfunction, wet or dry macular degeneration, proliferativevitreoretinopathy, vitreoretinopathy of any etiology, fibrosisassociated with ocular surgery such as treatment of glaucoma, retinalreattachment, cataract extraction, or drainage procedures of any kind,scarring in the cornea and conjunctiva, fibrosis in the cornealendothelium, alkali burn (e.g., alkali burn to the cornea),post-cataract surgery fibrosis of the lens capsule, excess scarring inthe tissue around the extraocular muscles in the strabismus surgery,anterior subcapsular cataract and posterior capsule opacification,anterior segment fibrotic diseases of the eye, fibrosis of the cornealstroma (e.g., associated with corneal opacification), fibrosis of thetrabecular network (e.g., associated with glaucoma), posterior segmentfibrotic diseases of the eye, fibrovascular scarring (e.g., in retinalor choroidal vasculature of the eye), retinal fibrosis, epiretinalfibrosis, retinal gliosis, subretinal fibrosis (e.g., associated withage related macular degeneration), fibrosis associated with post-retinaland glaucoma surgery, tractional retinal detachment in association withcontraction of the tissue in diabetic retinopathy.

Exemplary fibroproliferative diseases, conditions or disorders of theeye, fibrosis of the eye, ocular fibrosis or ophthalmic fibrosesinclude, but are not limited to, proliferative vitreoretinopathy,vitreoretinopathy of any etiology, fibrosis associated with retinaldysfunction, fibrosis associated with wet or dry macular degeneration,fibrosis associated with ocular surgery such as treatment of glaucoma,retinal reattachment, cataract extraction, or drainage procedures of anykind, scarring in the cornea and conjunctiva, fibrosis in the cornealendothelium, fibrosis associated with alkali burn, post-cataract surgeryfibrosis of the lens capsule, excess scarring the tissue around theextraocular muscles in the strabismus surgery, anterior subcapsularcataract and posterior capsule opacification, anterior segment fibroticdiseases of the eye, fibrosis of the corneal stroma (e.g., associatedwith corneal opacification), fibrosis of the trabecular network (e.g.,associated with glaucoma), posterior segment fibrotic diseases of theeye, fibrovascular scarring (e.g., in retinal or choroidal vasculatureof the eye), retinal fibrosis, epiretinal fibrosis, retinal gliosis,subretinal fibrosis (e.g., associated with age related maculardegeneration), fibrosis associated with post-retinal and glaucomasurgery, tractional retinal detachment in association with contractionof the tissue in diabetic retinopathy.

In various embodiments, the fibroproliferative disease, condition, ordisorders of the eye is selected from the group consisting ofproliferative vitreoretinopathy, fibrosis associated with ocularsurgery, post-cataract surgery fibrosis of the lens, fibrosis of thecorneal stroma and alkali burn.

An “immunoglobulin” or “native antibody” is a tetrameric glycoprotein.In a naturally-occurring immunoglobulin, each tetramer is composed oftwo identical pairs of polypeptide chains, each pair having one “light”(about 25 kDa) and one “heavy” chain (about 50-70 kDa). Theamino-terminal portion of each chain includes a variable region of about100 to 110 or more amino acids primarily responsible for antigenrecognition. The carboxy-terminal portion of each chain defines aconstant region primarily responsible for effector function. Human lightchains are classified as kappa (κ) and lambda (λ) light chains. Heavychains are classified as mu (μ), delta (Δ), gamma (γ), alpha (α), andepsilon (ε), and define the antibody's isotype as IgM, IgD, IgG, IgA,and IgE, respectively. Within light and heavy chains, the variable andconstant regions are joined by a “J” region of about 12 or more aminoacids, with the heavy chain also including a “D” region of about 10 moreamino acids. See generally, Fundamental Immunology, Ch. 7 (Paul, W.,ed., 2nd ed. Raven Press, N.Y. (1989)) (incorporated by reference in itsentirety for all purposes). The variable regions of each light/heavychain pair form the antibody binding site such that an intactimmunoglobulin has two binding sites.

Each heavy chain has at one end a variable domain (VH) followed by anumber of constant domains. Each light chain has a variable domain atone end (VL) and a constant domain at its other end; the constant domainof the light chain is aligned with the first constant domain of theheavy chain, and the light chain variable domain is aligned with thevariable domain of the heavy chain. Particular amino acid residues arebelieved to form an interface between the light and heavy chain variabledomains (Chothia et al., J. Mol. Biol. 196:901-917, 1987).

Immunoglobulin variable domains exhibit the same general structure ofrelatively conserved framework regions (FR) joined by threehypervariable regions or CDRs. From N-terminus to C-terminus, both lightand heavy chains comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3and FR4. The assignment of amino acids to each domain is in accordancewith the definitions of Kabat Sequences of Proteins of ImmunologicalInterest (National Institutes of Health, Bethesda, Md. (1987 and 1991)),or Chothia & Lesk, (J. Mol. Biol. 196:901-917, 1987); Chothia et al.,(Nature 342:878-883, 1989).

The hypervariable region of an antibody refers to the CDR amino acidresidues of an antibody which are responsible for antigen-binding. Thehypervariable region comprises amino acid residues from a CDR [e.g.,residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the light chainvariable domain and 31-35 (H1), 50-65 (H2) and 95-102 (H3) in the heavychain variable domain as described by Kabat et al., Sequences ofProteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991)] and/or thoseresidues from a hypervariable loop (e.g., residues 26-32 (L1), 50-52(L2) and 91-96 (L3) in the light chain variable domain and 26-32 (H1),53-55 (H2) and 96-101 (H3) in the heavy chain variable domain asdescribed by [Chothia et al., J. Mol. Biol. 196: 901-917 (1987)]. CDRshave also been identified and numbered according to ImMunoGenTics (IMGT)numbering (Lefranc, M.-P., The Immunologist, 7, 132-136 (1999); Lefranc,M.-P. et al., Dev. Comp. Immunol., 27, 55-77 (2003), which describes theCDR locations in the light and heavy chain variable domains as follows:CDR1, approximately residues 27 to 38; CDR2, approximately residues 56to 65; and, CDR3, approximately residues 105 to 116 (germline) orresidues 105 to 117 (rearranged). In one embodiment, it is contemplatedthat the CDRs are located at approximately residues 26-31 (L1), 49-51(L2) and 88-98 (L3) in the light chain variable domain and approximatelyresidues 26-33 (H1), 50-58 (H2) and 97-111 (H3) in the heavy chainvariable domain of an antibody heavy or light chain of approximatelysimilar length to those disclosed herein. However, one of skill in theart understands that the actual location of the CDR residues may varyfrom the projected residues described above when the sequence of theparticular antibody is identified.

Framework or FR residues are those variable domain residues other thanthe hypervariable region residues.

“Heavy chain variable region” as used herein refers to the region of theantibody molecule comprising at least one complementarity determiningregion (CDR) of said antibody heavy chain variable domain. The heavychain variable region may contain one, two, or three CDR of saidantibody heavy chain.

“Light chain variable region” as used herein refers to the region of anantibody molecule, comprising at least one complementarity determiningregion (CDR) of said antibody light chain variable domain. The lightchain variable region may contain one, two, or three CDR of saidantibody light chain, which may be either a kappa or lambda light chaindepending on the antibody.

The term “antibody” is used in the broadest sense and includes fullyassembled antibodies, tetrameric antibodies, monoclonal antibodies,polyclonal antibodies, multispecific antibodies (e.g., bispecificantibodies), antibody fragments that can bind an antigen (e.g., Fab′,F′(ab)2, Fv, single chain antibodies, diabodies), and recombinantpeptides comprising the forgoing as long as they exhibit the desiredbiological activity. An “immunoglobulin” or “tetrameric antibody” is atetrameric glycoprotein that consists of two heavy chains and two lightchains, each comprising a variable region and a constant region.Antigen-binding portions may be produced by recombinant DNA techniquesor by enzymatic or chemical cleavage of intact antibodies. Antibodyfragments or antigen-binding portions include, inter alia, Fab, Fab′,F(ab′)2, Fv, domain antibody (dAb), complementarity determining region(CDR) fragments, CDR-grafted antibodies, single-chain antibodies (scFv),single chain antibody fragments, chimeric antibodies, diabodies,triabodies, tetrabodies, minibody, linear antibody; chelatingrecombinant antibody, a tribody or bibody, an intrabody, a nanobody, asmall modular immunopharmaceutical (SMIP), a antigen-binding-domainimmunoglobulin fusion protein, a camelized antibody, a VHH containingantibody, or a variant or a derivative thereof, and polypeptides thatcontain at least a portion of an immunoglobulin that is sufficient toconfer specific antigen binding to the polypeptide, such as a one, two,three, four, five or six CDR sequences, as long as the antibody retainsthe desired biological activity.

“Monoclonal antibody” refers to an antibody obtained from a populationof substantially homogeneous antibodies, i.e., the individual antibodiescomprising the population are identical except for possible naturallyoccurring mutations that may be present in minor amounts.

“Antibody variant” as used herein refers to an antibody polypeptidesequence that contains at least one amino acid substitution, deletion,or insertion in the variable region of the reference antibody variableregion domains. Variants may be substantially homologous orsubstantially identical to the unmodified antibody.

A “chimeric antibody,” as used herein, refers to an antibody containingsequence derived from two different antibodies (see, e.g., U.S. Pat. No.4,816,567) which typically originate from different species. Mosttypically, chimeric antibodies comprise human and rodent antibodyfragments, generally human constant and mouse variable regions.

A “neutralizing antibody” is an antibody molecule which is able toeliminate or significantly reduce a biological function of a targetantigen to which it binds. Accordingly, a “neutralizing” anti-targetantibody is capable of eliminating or significantly reducing abiological function, such as enzyme activity, ligand binding, orintracellular signaling.

An “isolated” antibody is one that has been identified and separated andrecovered from a component of its natural environment. Contaminantcomponents of its natural environment are materials that would interferewith diagnostic or therapeutic uses for the antibody, and may includeenzymes, hormones, and other proteinaceous or non-proteinaceous solutes.In preferred embodiments, the antibody will be purified (1) to greaterthan 95% by weight of antibody as determined by the Lowry method, andmost preferably more than 99% by weight, (2) to a degree sufficient toobtain at least 15 residues of N-terminal or internal amino acidsequence by use of a spinning cup sequenator, or (3) to homogeneity bySDS-PAGE under reducing or nonreducing conditions using Coomassie blueor, preferably, silver stain. Isolated antibody includes the antibody insitu within recombinant cells since at least one component of theantibody's natural environment will not be present. Ordinarily, however,isolated antibody will be prepared by at least one purification step.

As used herein, an antibody that “specifically binds” is “targetspecific”, is “specific for” target or is “immunoreactive” with thetarget antigen refers to an antibody or antibody substance that bindsthe target antigen with greater affinity than with similar antigens. Inone aspect of the disclosure, the target-binding polypeptides, orfragments, variants, or derivatives thereof, will bind with a greateraffinity to human target as compared to its binding affinity to targetof other, i.e., non-human, species, but binding polypeptides thatrecognize and bind orthologs of the target are within the scopeprovided.

For example, a polypeptide that is an antibody or fragment thereof“specific for” its cognate antigen indicates that the variable regionsof the antibodies recognize and bind the polypeptide of interest with adetectable preference (i.e., able to distinguish the polypeptide ofinterest from other known polypeptides of the same family, by virtue ofmeasurable differences in binding affinity, despite the possibleexistence of localized sequence identity, homology, or similaritybetween family members). It will be understood that specific antibodiesmay also interact with other proteins (for example, S. aureus protein Aor other antibodies in ELISA techniques) through interactions withsequences outside the variable region of the antibodies, and inparticular, in the constant region of the molecule. Screening assays todetermine binding specificity of an antibody for use in the methods ofthe present disclosure are well known and routinely practiced in theart. For a comprehensive discussion of such assays, see Harlow et al.(Eds), Antibodies A Laboratory Manual; Cold Spring Harbor Laboratory;Cold Spring Harbor, N.Y. (1988), Chapter 6. Antibodies for use in themethods can be produced using any method known in the art.

The term “epitope” refers to that portion of any molecule capable ofbeing recognized by and bound by a selective binding agent at one ormore of the antigen binding regions. Epitopes usually consist ofchemically active surface groupings of molecules, such as, amino acidsor carbohydrate side chains, and have specific three-dimensionalstructural characteristics as well as specific charge characteristics.Epitopes as used herein may be contiguous or non-contiguous. Moreover,epitopes may be mimetic (mimotopes) in that they comprise a threedimensional structure that is identical to the epitope used to generatethe antibody, yet comprise none or only some of the amino acid residuesfound in the target that were used to stimulate the antibody immuneresponse. As used herein, a mimotope is not considered a differentantigen from the epitope bound by the selective binding agent; theselective binding agent recognizes the same three-dimensional structureof the epitope and mimotope.

The term “derivative” when used in connection with antibody substancesand polypeptides of the present disclosure refers to polypeptideschemically modified by such techniques as ubiquitination, conjugation totherapeutic or diagnostic agents, labeling (e.g., with radionuclides orvarious enzymes), covalent polymer attachment such as pegylation(derivatization with polyethylene glycol) and insertion or substitutionby chemical synthesis of amino acids such as ornithine, which do notnormally occur in human proteins. Derivatives retain the bindingproperties of underivatized molecules of the disclosure.

“Detectable moiety” or a “label” refers to a composition detectable byspectroscopic, photochemical, biochemical, immunochemical, or chemicalmeans. For example, useful labels include 32P, 35S, fluorescent dyes,electron-dense reagents, enzymes (e.g., as commonly used in an ELISA),biotin-streptavadin, dioxigenin, haptens and proteins for which antiseraor monoclonal antibodies are available, or nucleic acid molecules with asequence complementary to a target. The detectable moiety oftengenerates a measurable signal, such as a radioactive, chromogenic, orfluorescent signal, that can be used to quantitate the amount of bounddetectable moiety in a sample.

The term “therapeutically effective amount” is used herein to indicatethe amount of target-specific composition of the disclosure that iseffective to ameliorate or lessen symptoms or signs of diseaseassociated with target protein expression.

The terms “treat”, “treating” and “treatment”, as used with respect tomethods herein refer to eliminating, reducing, suppressing orameliorating, either temporarily or permanently, either partially orcompletely, a clinical symptom, manifestation or progression of anevent, disease or condition associated with TGFβ expression. Suchtreating need not be absolute to be useful.

The present disclosure provides a target-specific antibody, which maycomprise those exemplary sequences set out in Table 1 (FIG. 23),fragments, variants and derivatives thereof, pharmaceutical formulationsincluding a target-specific antibody recited above, methods of preparingthe pharmaceutical formulations, and methods of treating patients withthe pharmaceutical formulations and compounds.

Depending on the amino acid sequence of the constant domain of theirheavy chains, immunoglobulins can be assigned to different classes, IgA,IgD, IgE, IgG and IgM, which may be further divided into subclasses orisotypes, e.g. IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. The subunitstructures and three-dimensional configurations of different classes ofimmunoglobulins are well known. Different isotypes have differenteffector functions; for example, IgG1 and IgG3 isotypes have ADCCactivity. An antibody disclosed herein, if it comprises a constantdomain, may be of any of these subclasses or isotypes.

The antibodies of the present disclosure may exhibit binding affinity toone or more TGFβ antigens of a Kd of less than or equal to about 10⁻⁵ M,less than or equal to about 10⁻⁶ M, or less than or equal to about 10⁻⁷M, or less than or equal to about 10⁻⁸ M, or less than or equal to about10⁻⁹ M, 10⁻¹⁰ M, 10⁻¹¹ M, or 10⁻¹² M or less. Such affinities may bereadily determined using conventional techniques, such as by equilibriumdialysis; by using surface plasmon resonance (SPR) technology (e.g., theBIAcore 2000 instrument, using general procedures outlined by themanufacturer); by radioimmunoassay using 125I labeled target antigen; orby another method set forth in the examples below or known to theskilled artisan. The affinity data may be analyzed, for example, by themethod of Scatchard et al., (Ann N.Y. Acad. Sci., 51:660, 1949).

A KinExA kinetic exclusion assay is also useful to measure the affinityof an antibody for its antigen. KinExA technology measures bindingevents in the solution phase, rather than binding events between asolution phase and a solid phase. In addition, while many methods formeasuring binding events require at least one reactant be modifiedthrough immobilization or labeling, the KinExA method does not requiremodification of molecules under study. The KinExA method is believed toallow a wider range of binding constants to be measured than othermethods currently available. Additional description about KinExA devicesand operation for antibody characterization is available from themanufacturer (Sapidyne Instruments, Inc., Boise, Id.) and can be foundin the published literature, for example U.S. Pat. No. 6,664,114 andDarling et al., “Kinetic Exclusion Assay Technology: Characterization ofMolecular Interactions.” Assay and Drug Development Technologies, 2004,2:647-657.

Transforming Growth Factor β

TGFβ is a disulfide linked dimer that is synthesized as a preproproteinof about 400 amino acids (aa) which is cleaved prior to secretion toproduce mature TGFβ. The N-terminal cleavage fragment, known as the“latency-associated peptide” (LAP), may remain noncovalently bound tothe dimer, thereby inactivating TGFβ. TGFβ isolated in vivo, is foundpredominantly in the inactive, “latent” form, i.e., associated with LAP.Latent TGFβ complex may be activated in several ways, for example, bybinding to a cell surface receptor called the cation-independentmannose-6-phosphate/insulin-like growth factor II receptor. Bindingoccurs through mannose-6-phosphate residues attached at glycosylationsites within LAP. Upon binding to the receptor, TGFβ is released in itsmature form. Mature, active TGFβ is then free to bind to its receptorand exert its biological functions. The major TGFβ binding domain in thetype II TGFβ receptor has been mapped to a 19 amino acid sequence(Demetriou et al., J. Biol. Chem., 271:12755, 1996). See also U.S. Pat.No. 7,867,496.

Currently, there are five known isoforms of TGFβ (TGFβ1 to TGFβ5;TGFβ1-3 are mammalian, TGFβ4 is found in chicken; and TGFβ5 found infrog), all of which are homologous among each other (60-80% identity),form homodimers of about 25 kDa, and act upon common TGFβ receptors(TGFβ-RI, TGFβ-RII, TGFβ-RIIB, and TGFβ-RIII). The structural andfunctional aspects of TGFβ as well as TGFβ receptors are well-known inthe art (see, for example, Cytokine Reference, eds. Oppenheim et al.,Academic Press, San Diego, Calif., 2001). TGFβ is well-conserved amongspecies. For example, the amino acid sequences of rat and human matureTGFβ1s are nearly identical. See also U.S. Pat. No. 7,867,496.

TGFβ1 plays an important role in the process of wound healing inbiological tissues (New Engl. J. Med., Vol. 331, p. 1286, 1994 and J.Cell. Biol., Vol. 119, p. 1017,1992). At the site of wounded tissue,biological reactions such as infiltration of inflammatory cells andfibroblast cells, production of extracellular matrix (ECM) andvascularization, and cell growth for the subsequent tissue regenerationoccur to repair the injured tissue. See also U.S. Pat. No. 7,579,186.

TGFβ2 deficient mice demonstrate significant developmental defects,including heart, lung, craniofacial, limb, spine, eye, ear andurogenital defects (Dunker et al., Eur J Biol 267:6982-8, 2001). TGFβ3deficient mice demonstrate almost 100% lethality by 24 hrs after birth.These mice show significant palate impairment and delayed pulmonarydevelopment (Dunker et al., supra). TGFβ2 has also been implicated inthe development of glaucoma (Luthen-Driscoll, Experimental Eye Res81:1-4, 2005), fibrosis associated with Crohn's Disease (Van Assche etal., Inflamm Bowel Dis. 10:55-60, 2004), in wound healing and diabeticnephropathy (Pohlers et al., Biochim Biophys Acta 1792:746-56, 2009)

It has been observed that many human tumors (deMartin et al., EMBO J.,6: 3673 (1987), Kuppner et al., Int. J. Cancer, 42: 562 (1988)) and manytumor cell lines (Derynck et al., Cancer Res., 47: 707 (1987), Robertset al., Br. J. Cancer, 57: 594 (1988)) produce TGFβ and suggests apossible mechanism for those tumors to evade normal immunologicalsurveillance.

TGFβ isoform expression in cancer is complex and variable with differentcombinations of TGFβ isoforms having different roles in particularcancers. See e.g., U.S. Pat. No. 7,927,593. For example, TGFβ1 and TGFβ3may play a greater role in ovarian cancer and its progression thanTGFβ2; while TGFβ1 and TGFβ2 expression is greater in higher gradechondrosarcoma tumors than TGFβ3. In human breast cancer, TGFβ1 andTGFβ3 are highly expressed, with TGFβ3 expression appearing to correlatewith overall survival—patients with node metastasis and positive TGFβ3expression have poor prognostic outcomes. However, in colon cancer,TGFβ1 and TGFβ2 are more highly expressed than TGFβ3 and are present atgreater circulating levels than in cancer-free individuals. In gliomas,TGFβ2 is important for cell migration.

TGFβ expression has also been implicated in the onset of various tissuefibroses, such as nephrosclerosis, pulmonary fibrosis and cirrhosis; aswell as the onset of various states, such as chronic hepatitis,rheumatoid arthritis, vascular restenosis, and keloid of skin. Fibrosescontemplated, including fibroses associated with a disease or disorder(e.g., fibroproliferative diseases or disorders), or treatment of adisease or disorder, include, but are not limited to, pulmonaryfibrosis, idiopathic pulmonary fibrosis, peribronchiolar fibrosis,interstitial lung disease, chronic obstructive pulmonary disease (COPD),small airway disease (e.g., obstructive bronchiolitis), emphysema, adultor acute respiratory distress syndrome (ARDS), acute lung injury (ALI);pulmonary fibrosis due to infectious or toxic agents, kidney fibrosis,glomerulonephritis (GN) of all etiologies, e.g., mesangial proliferativeGN, immune GN, and crescentic GN, glomerulosclerosis, tubulointerstitialinjury, renal interstitial fibrosis, renal fibrosis and all causes ofrenal interstitial fibrosis, renal fibrosis resulting from complicationsof drug exposure, including cyclosporin treatment of transplantrecipients, e.g. cyclosporin treatment, HIV-associated nephropathy;transplant necropathy, diabetic kidney disease (e.g., diabeticnephropathy), nephrogenic systemic fibrosis, diabetes, idiopathicretroperitoneal fibrosis, scleroderma, liver fibrosis, hepatic diseasesassociated with excessive scarring and progressive sclerosis, includingliver cirrhosis due to all etiologies, disorders of the biliary tree,hepatic dysfunction attributable to infections, fibrocystic diseases,cardiovascular diseases, such as congestive heart failure; dilatedcardiomyopathy, myocarditis, vascular stenosis, cardiac fibrosis (e.g.,post-infarction cardiac fibrosis), post myocardial infarction, leftventricular hypertrophy, veno-occlusive disease, restenosis (e.g.,post-angioplasty restenosis), arteriovenous graft failure,atherosclerosis, hypertension, hypertensive heart disease, cardiachypertrophy, hypertrophic cardiomyopathy, heart failure, disease of theaorta, progressive systemic sclerosis; polymyositis, systemic lupuserythematosus, dermatomyositis, fascists, Raynaud's syndrome, rheumatoidarthritis, proliferative vitreoretinopathy, vitreoretinopathy of anyetiology, fibrosis associated with ocular surgery such as treatment ofglaucoma, fibrosis associated with retinal dysfunction, retinalreattachment, cataract extraction or drainage procedures of any kind,scarring in the cornea and conjunctiva, fibrosis in the cornealendothelium, fibrosis associated with alkali burn, post-cataract surgeryfibrosis of the lens capsule, excess scarring the tissue around theextraocular muscles in the strabismus surgery, anterior subcapsularcataract and posterior capsule opacification, anterior segment fibroticdiseases of the eye, fibrosis of the corneal stroma (e.g., associatedwith corneal opacification), fibrosis of the trabecular network (e.g.,associated with glaucoma), posterior segment fibrotic diseases of theeye, fibrovascular scarring (e.g., in retinal or choroidal vasculatureof the eye), retinal fibrosis, epiretinal fibrosis, retinal gliosis,subretinal fibrosis (e.g., associated with age related maculardegeneration), post-retinal and glaucoma surgery, tractional retinaldetachment in association with contraction of the tissue in diabeticretinopathy, Peyronie's disease, systemic sclerosis, post-spinal cordinjury, osteoporosis, Camurati-Engelmann disease, Crohn's disease,scarring, Marfan syndrome, premature ovarian failure, Alzheimer'sDisease and Parkinson's Disease, fibrosis due to surgical incisions ormechanical trauma, fibrosis associated with ocular surgery; andexcessive or hypertrophic scar or keloid formation in the dermisoccurring during wound healing resulting from trauma or surgical wounds.

In pulmonary fibrosis and nephrosclerosis, the concentration of TGFβ ishigh and leads to the progress of the morbid states, such as fibrosis(Yamamoto et al., Kidney Int. 45:916-27, 1994 and Westergren-Thorsson etal., J. Clin. Invest. 92:632-7, 1993). The persistent tissue injury hasbeen presumed to continuously transduce signals to express TGFβ, tosuppress the negative regulation signal for TGFβ expression by ECM, orcause both events synergistically in pulmonary fibrosis andnephrosclerosis. Suppressing TGFβ activity and extracellular matrixaccumulation in diagnosis and treatment of fibrotic diseases, using ainhibitor of TGFβ is disclosed in WO 1991/04748, WO 1993/10808 and WO2000/40227. Neutralizing anti-TGF-beta antibodies have been used in thetreatment of experimental diabetic kidney disease (Han and Ziyadeh,Peritoneal dialysis international, 19 Suppl 2: S234-237 (1999)). Seealso U.S. Pat. No. 7,527,791 further describing use of inhibitors ofTGFβ in various indications, hereby incorporated by reference.

Exemplary eye diseases (e.g., ocular, optic, ophthalmic orophthalmological diseases), conditions or disorders, include but are notlimited to, fibroproliferative disorders, fibrosis of the eye,ophthalmic fibroses, retinal dysfunction, fibrosis associated withretinal dysfunction, wet or dry macular degeneration, proliferativevitreoretinopathy, vitreoretinopathy of any etiology, fibrosisassociated with ocular surgery such as treatment of glaucoma, retinalreattachment, cataract extraction, or drainage procedures of any kind,scarring in the cornea and conjunctiva, fibrosis in the cornealendothelium, alkali burn (e.g., alkali burn to the cornea),post-cataract surgery fibrosis of the lens capsule, excess scarring inthe tissue around the extraocular muscles in the strabismus surgery,anterior subcapsular cataract and posterior capsule opacification,anterior segment fibrotic diseases of the eye, fibrosis of the cornealstroma (e.g., associated with corneal opacification), fibrosis of thetrabecular network (e.g., associated with glaucoma), posterior segmentfibrotic diseases of the eye, fibrovascular scarring (e.g., in retinalor choroidal vasculature of the eye), retinal fibrosis, epiretinalfibrosis, retinal gliosis, subretinal fibrosis (e.g., associated withage related macular degeneration), fibrosis associated with post-retinaland glaucoma surgery, tractional retinal detachment in association withcontraction of the tissue in diabetic retinopathy.

Exemplary fibroproliferative diseases, conditions or disorders of theeye, fibrosis of the eye, ocular fibrosis or ophthalmic fibrosesinclude, but are not limited to, proliferative vitreoretinopathy,vitreoretinopathy of any etiology, fibrosis associated with retinaldysfunction, fibrosis associated with wet or dry macular degeneration,fibrosis associated with ocular surgery such as treatment of glaucoma,retinal reattachment, cataract extraction, or drainage procedures of anykind, scarring in the cornea and conjunctiva, fibrosis in the cornealendothelium, fibrosis associated with alkali burn, post-cataract surgeryfibrosis of the lens capsule, excess scarring the tissue around theextraocular muscles in the strabismus surgery, anterior subcapsularcataract and posterior capsule opacification, anterior segment fibroticdiseases of the eye, fibrosis of the corneal stroma (e.g., associatedwith corneal opacification), fibrosis of the trabecular network (e.g.,associated with glaucoma), posterior segment fibrotic diseases of theeye, fibrovascular scarring (e.g., in retinal or choroidal vasculatureof the eye), retinal fibrosis, epiretinal fibrosis, retinal gliosis,subretinal fibrosis (e.g., associated with age related maculardegeneration), fibrosis associated with post-retinal and glaucomasurgery, tractional retinal detachment in association with contractionof the tissue in diabetic retinopathy.

In various embodiments, the fibroproliferative disease, condition, ordisorders of the eye is selected from the group consisting ofproliferative vitreoretinopathy, fibrosis associated with ocularsurgery, post-cataract surgery fibrosis of the lens, fibrosis of thecorneal stroma and alkali burn.

Antibody Polypeptides

The present disclosure encompasses amino acid molecules encoding targetspecific antibodies. In exemplary embodiments, a target specificantibody of the disclosure can comprise a human kappa (κ) or a humanlambda (λ) light chain or an amino acid sequence derived therefrom, or ahuman heavy chain or a sequence derived therefrom, or both heavy andlight chains together in a single chain, dimeric, tetrameric or otherform. In some embodiments, a heavy chain and a light chain of a targetspecific immunoglobulin are different amino acid molecules. In otherembodiments, the same amino acid molecule contains a heavy chainvariable region and a light chain variable region of a target specificantibody.

In some embodiments, the amino acid sequence of the human anti-targetantibody comprises one or more CDRs of the amino acid sequence of themature (i.e., missing signal sequence) light chain variable region (VL)of antibodies XPA.42.068, XPA.42.089 and XPA.42.681 set out in Table 1or SEQ ID NOs: 4,8 and 12 or variants thereof, including CDR grafted,modified, humanized, chimeric, or Human Engineered antibodies or anyother variants described herein. In some embodiments, the VL comprisesthe amino acid sequence from the beginning of the CDR1 to the end of theCDR3 of the light chain of any one of the foregoing antibodies.

In one embodiment, the target specific antibody comprises a light chainCDR1, CDR2 or CDR3 ((LCDR1, LCDR2, LCDR3), each of which areindependently selected from the CDR1, CDR2 and CDR3 regions of anantibody having a light chain variable region comprising the amino acidsequence of the VL region set out in SEQ ID NOs: 4,8 and 12, a nucleicacid encoding the VH region set out in SEQ ID NOs: 4, 8, and 12, orencoded by a nucleic acid molecule encoding the VL region set out in SEQID NOs: 3, 7, and 11. In one embodiment, the light chain CDR1 is fromapproximately residues 24-34, CDR2 is from approximately residues 50-56and CDR3 extends from approximately residues 89-97, according to Chothianumbering. In an alternate embodiment, it is contemplated that the heavychain CDRs are located at approximately residues 27 to 38 (CDR1);approximately residues 56 to 65 (CDR2); and, approximately residues 105to 116 (germline) or residues 105 to 117 (CDR3) according toImMunoGenTics (IMGT) numbering. In one embodiment, it is contemplatedthat the light chain CDRs are located at approximately residues 26-31(L1), 49-51 (L2) and 88-97 (L3) in the light chain variable domain of anantibody light chain of approximately similar length to those disclosedherein. A polypeptide of the target specific antibody may comprise theCDR1, CDR2 and CDR3 regions of an antibody comprising the amino acidsequence of the VL region selected from the group consisting ofXPA.42.068, XPA.42.089 and XPA.42.681.

In some embodiments, the human target specific antibody comprises one ormore CDRs of the amino acid sequence of the mature (i.e., missing signalsequence) heavy chain variable region (VH) of antibody XPA.42.068,XPA.42.089 and XPA.42.681 set out in Table 1 or SEQ ID NOs: 2, 6 and 10or variants thereof. In some embodiments, the VH comprises the aminoacid sequence from the beginning of the CDR1 to the end of the CDR3 ofany one of the heavy chain of the foregoing antibodies.

In one embodiment, the target specific antibody comprises a heavy chainCDR1, CDR2 or CDR3 (HCDR1, HCDR2, HCDR3), each of which areindependently selected from the CDR1, CDR2 and CDR3 regions of anantibody having a heavy chain variable region comprising the amino acidsequence of the VH region set out in SEQ ID NOs: 2, 6, and 10, a nucleicacid encoding the VH region set out in SEQ ID NOs: 2, 6, and 10, orencoded by a nucleic acid molecule encoding the VH region set out in SEQID NOs: 1, 5, and 9. It is further contemplated that a target specificantibody comprises a heavy chain CDR1, CDR2 or CDR3, each of which areindependently selected from the CDR1, CDR2 and CDR3 regions of anantibody having a heavy chain variable region comprising the amino acidsequence of the VH region set out in SEQ ID NOs: 2, 6, and 10. In oneembodiment, the heavy chain CDRs are located according to Chothianumbering: CDR1 is from approximately residues 26-35, CDR2 is fromapproximately residues 50-58 and CDR3 extends from approximatelyresidues 95-102 (or 95-111 or 95-118). In an alternate embodiment, it iscontemplated that the heavy chain CDRs are located at CDR1,approximately residues 27 to 38 (CDR1); approximately residues 56 to 65(CDR2); and, CDR3, approximately residues 105 to 116 (germline) orresidues 105 to 117 CDR3) according to ImMunoGenTics (IMGT) numbering.In one embodiment, it is contemplated that the heavy chain CDRs arelocated at approximately residues 26-33 (H1), 50-58 (H2) and 97-111 (H3)in the heavy chain variable domain of an antibody heavy chain ofapproximately similar length to those disclosed herein. A polypeptide ofthe target specific antibody may comprise the CDR1, CDR2 and CDR3regions of an antibody comprising the amino acid sequence of the VHregion selected from the group consisting of XPA.42.068, XPA.42.089 andXPA.42.681.

In another embodiment, the antibody comprises a mature light chainvariable region as disclosed above and a mature heavy chain variableregion as disclosed above, optionally paired as set forth in Table 1(FIG. 23).

In exemplary embodiments, the disclosure contemplates:

a monoclonal antibody that retains any one, two, three, four, five, orsix of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, or LCDR3 of any one of SEQ IDNOs: 13, 19 and 25; 14, 20 and 26; 15, 21 and 27 and SEQ ID NOs: 16, 22and 28; 17, 23 and 29; and 18, 24 and 30, respectively, optionallyincluding one or two mutations in any of such CDR(s), e.g., aconservative or non-conservative substitution, and optionally paired asset forth in Table 1;

a monoclonal antibody that retains all of HCDR1, HCDR2, HCDR3, or theheavy chain variable region of any one of SEQ ID NOs: 13, 19 and 25; 14,20 and 26; and 15, 21 and 27, optionally including one or two mutationsin any of such CDR(s), optionally further comprising any suitable heavychain constant region, e.g., IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, orIgE, a human sequence thereof, or a hybrid thereof;

a monoclonal antibody that retains all of LCDR1, LCDR2, LCDR3, or thelight chain variable region of any one SEQ ID NOs: 16, 22 and 28; 17, 23and 29; and 18, 24 and 30, optionally including one or two mutations inany of such CDR(s), optionally further comprising any suitable lightchain constant region, e.g., a kappa or lambda light chain constantregion, a human sequence thereof, or a hybrid thereof.

In some embodiments, the antibody comprises all three light chain CDRs,all three heavy chain CDRs, or all six CDRs of the light and heavychain, paired as set forth in Table 1. In some exemplary embodiments,two light chain CDRs from an antibody may be combined with a third lightchain CDR from a different antibody. Alternatively, a LCDR1 from oneantibody can be combined with a LCDR2 from a different antibody and aLCDR3 from yet another antibody, particularly where the CDRs are highlyhomologous. Similarly, two heavy chain CDRs from an antibody may becombined with a third heavy chain CDR from a different antibody; or aHCDR1 from one antibody can be combined with a HCDR2 from a differentantibody and a HCDR3 from yet another antibody, particularly where theCDRs are highly homologous.

In some embodiments, an antibody is provided that comprises apolypeptide having an amino acid sequence at least about 65%, 70%, 75%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96% , 97%, 98%, 99% or more identical to the heavy chainvariable region set out in SEQ ID NOs: 2, 6, and 10 and/or an amino acidsequence an amino acid sequence at least about 65%, 70%, 75%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96% , 97%, 98%, 99% or more identical to the light chain variable regionset out in SEQ ID NOs: 4,8 and 12, the antibody further comprising atleast one, two, three, four, five or all of HCDR1, HCDR2, HCDR3, LCDR1,LCDR2 or LCDR3. In some embodiments, the amino acid sequence withpercentage identity to the light chain variable region may comprise one,two or three of the light chain CDRs. In other embodiments, the aminoacid sequence with percentage identity to the heavy chain variableregion may comprise one, two, or three of the heavy chain CDRs.

In another embodiment, an antibody is provided that comprises apolypeptide having an amino acid sequence at least about 65%, 70%, 75%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96% , 97%, 98%, 99% or more identical to all three HCDRs inthe heavy chain variable region of an antibody sequence in Table 1, theCDRs set out in SEQ ID NOs: 13, 19 and 25; 14, 20 and 26; and 15, 21 and27.

In a related embodiment, an antibody is provided that comprises apolypeptide having an amino acid sequence at least about 65%, 70%, 75%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96% , 97%, 98%, 99% or more identical to the all three LCDRsin the light chain variable region of an antibody sequence in Table 1,the CDRs set out in SEQ ID NOs: 16, 22 and 28; 17, 23 and 29; and 18, 24and 30.

In a further embodiment, an antibody is provided that comprises apolypeptide having an amino acid sequence at least about 65%, 70%, 75%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96% , 97%, 98%, 99% or more identical to the all six CDRs inthe heavy chain and light chain variable regions of an antibody sequencein Table 1, the CDRs set out in SEQ ID NOs: 13, 19 and 25; 14, 20 and26; and 15, 21 27; 16, 22 and 28; 17, 23 and 29; and 18, 24 and 30.

It is contemplated that the antibodies of the disclosure may have one,or two or more amino acid substitutions in the CDR regions of theantibody, e.g., non-conservative or conservative substitutions.

In a related embodiment, the residues of the framework are altered. Theheavy chain framework regions which can be altered lie within regionsdesignated H-FR1, H-FR2, H-FR3 and H-FR4, which surround the heavy chainCDR residues, and the residues of the light chain framework regionswhich can be altered lie within the regions designated L-FR1, L-FR2,L-FR3 and L-FR4, which surround the light chain CDR residues. An aminoacid within the framework region may be replaced, for example, with anysuitable amino acid identified in a human framework or human consensusframework.

In exemplary embodiments, an anti-TGFβ antibody described hereinspecifically binds at least one isoform of TGFβ selected from the groupconsisting of TGFβ1, TGFβ2, and TGFβ3. In other embodiments, theanti-TGFβ antibody specifically binds: (a) TGFβ1, TGFβ2, and TGFβ3(“pan-reactive antibody” or “pan-binding antibody”); (b) TGFβ1 andTGFβ2; (c) TGFβ1 and TGFβ3; and (d) TGFβ2 and TGFβ3. In exemplaryembodiments, an anti-TGFβ antibody described herein binds at least oneisoform of TGFβ with an affinity of 10⁻⁶ M, 10⁻⁷ M, 10⁻⁸ M, 10⁻⁹ M,10⁻¹⁰ M, 10⁻¹¹ M, or 10⁻¹² M or less (lower meaning higher bindingaffinity), or optionally binds two TGFβ isoforms, or all of TGFβ1, 2, or3 with an affinity of 10⁻⁶ M. 10⁻⁷ M, 10⁻⁸ M, 10⁻⁹ M 10⁻¹⁰ M, 10⁻¹¹ M,or 10⁻¹² M or less for one or more of the isoforms. In otherembodiments, an antibody described herein binds to TGFβ1 and TGFβ2 withat least 2-50 fold, 10-100 fold, 2-fold, 5-fold, 10-fold, 25-fold,50-fold or 100-fold, or 20-50%, 50-100%, 20%, 25%, 30%, 40%, 50%, 60%,70%, 80%, 90% or 100% higher affinity (e.g., preferentially binds toTGFβ1 and TGFβ2) compared to binding to TGFβ3. Alternatively, anantibody described herein, binds each of TGFβ isoforms TGFβ1, TGFβ2 andTGFβ3 with an affinity within 3-fold, 5-fold or 10-fold of each other.

In some embodiments, antibody neutralization of TGFβ1 and TGFβ2 is atleast 2-50 fold, 10-100 fold, 2-fold, 5-fold, 10-fold, 25-fold, 50-foldor 100-fold, or 20-50%, 50-100%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%,90% or 100% more potent that neutralization of TGFβ3.

Heavy and light chain amino acid sequences of XPA.42.089 are set out inSEQ ID NOs: 6 and 8, respectively. Heavy and light chain amino acidsequences of XPA.42.068 are set out in SEQ ID NOs: 2 and 4,respectively, and heavy and light chain amino acid sequences ofXPA.42.681 are set out in SEQ ID NOs: 10 and 12, respectively.

Antibody Nucleic Acids

The present disclosure also encompasses nucleic acid molecules encodingtarget specific antibodies. In some embodiments, different nucleic acidmolecules encode a heavy chain variable region and a light chainvariable region of a target specific antibody. In other embodiments, thesame nucleic acid molecule encodes a heavy chain and a light chainvariable regions of a target specific antibody. In one embodiment, thenucleic acid encodes a target specific antibody of the presentdisclosure, as well as any of the polypeptides encoded by the nucleicacids described herein.

In one aspect, a nucleic acid molecule of the present disclosurecomprises a nucleotide sequence that encodes the VL amino acid sequenceof antibodies XPA.42.068, XPA.42.089 and XPA.42.681 set out in SEQ IDNOs: 4, 8 and 12 or a portion thereof. In a related aspect, the VL aminoacid sequence is a consensus sequence. In some embodiments, the nucleicacid encodes the amino acid sequence of the light chain CDRs of saidantibody. In some embodiments, said portion is a contiguous portioncomprising CDR1-CDR3. In one embodiment, said portion comprises at leastone, two or three of a light chain CDR1, CDR2, or CDR3 region,optionally with a different human or human consensus framework, andoptionally with 1, or up to 2, or up to 3 mutations in the collective 3CDRs.

In one embodiment the present disclosure provides antigen-bindingcompounds, including functional fragments, having a variable regionamino acid sequence set forth in any one of SEQ ID NOs: 2, 6, and 10 and4, 8 and 12. In a related embodiment, an aforementioned antigen bindingcompound is selected from the group consisting of a fully assembledtetrameric antibody, a monoclonal antibody a humanized antibody; a humanantibody; a chimeric antibody; a multispecific antibody, an antibodyfragment, Fab, F(ab′)2; Fv; scFv or single-chain antibody fragment; adiabody; triabody, tetrabody, minibody, linear antibody; chelatingrecombinant antibody, a tribody or bibody, an intrabody, a nanobody, asmall modular immunopharmaceutical (SMIP), a binding-domainimmunoglobulin fusion protein, a camelized antibody, a VHH containingantibody, or a variant or derivative of any one of these antibodies,that comprise one or more CDR sequences of the disclosure and exhibitthe desired biological activity, or a mixture of two or more antibodies.The antigen binding compounds of the present disclosure preferablyretain binding affinity of 10⁻⁶, 10⁻⁷, 10⁻⁸, 10⁻⁹, 10⁻¹⁰, 10⁻¹¹ M orless for one or more of TGFβ1, TGFβ2 and TGFβ3, as measured by surfaceplasmon resonance.

In one aspect, the antibodies of the present disclosure comprise a heavychain variable region or light chain variable region as set out in aminoacid sequences SEQ ID NOs: 2, 6, and 10 and SEQ ID NOs: 4, 8 and 12,respectively, as paired in Table 1. It is further contemplated that theantibodies may comprise all or part of the antibodies set out in theabove amino acid sequences. In one embodiment, the antibodies compriseat least one of CDR1, CDR2, or CDR3 of the heavy chain of SEQ ID NOs: 2,6, and 10, or at least one of CDR1, CDR2 or CDR3 of the light chain ofSEQ ID NOs: 4, 8 and 12, as paired in Table 1.

In one embodiment, the heavy chain comprises an amino acid sequenceidentified as a heavy chain CDR3 sequence. Such a “heavy chain CDR3sequence” (HCDR3) includes an amino acid sequence identified as a heavychain CDR3 sequence set out in Table 1 and SEQ ID NOs: 15, 21 and 27.Alternatively, the HCDR3 sequence comprises an amino acid sequence thatcontains one or more amino acid changes (e.g., substitution, insertionor deletion) compared to any HCDR3 amino acid sequence identified inTable 1. Preferable substitutions include a substitution to an aminoacid at the corresponding position within another HCDR3 of Table 1.Alternatively, the HCDR3 sequence may comprise a consensus amino acidsequence of the HCDR3 described herein.

The heavy chain comprising a HCDR3 sequence described above may furthercomprise a “heavy chain CDR1 sequence” (HCDR1), which includes any ofthe amino acid sequences identified as an HCDR1 in SEQ ID NOs: 13, 19and 25 and Table 1, amino acid sequences that contain one or more aminoacid changes compared to any HCDR1 identified in SEQ ID NOs: 13, 19 and25 and Table 1, preferably a substitution to an amino acid at thecorresponding position within another HCDR1 of Table 1, or a consensussequence of the HCDR1 described herein.

Alternatively, the heavy chain comprising a HCDR3 sequence describedabove may further comprise a “heavy chain CDR2 sequence” (HCDR2), whichincludes any of the amino acid sequences identified as an HCDR2 in SEQID NOs: 14, 20 and 26 and Table 1, amino acid sequences that contain oneor more amino acid changes compared to any HCDR2 identified in SEQ IDNOs: 14, 20 and 26 and Table 1, preferably a substitution to an aminoacid at the corresponding position within another HCDR2 of Table 1, or aconsensus sequence of the HCDR2 described herein.

The heavy chain comprising a heavy chain CDR3 sequence described abovemay also comprise both (a) a heavy chain CDR1 sequence described aboveand (b) a heavy chain CDR2 sequence of the invention described above.

One aspect of the present disclosure provides an antibody that bindstarget antigen comprising a heavy chain that comprises any one, two,and/or three of the heavy chain CDR sequences described below.

Any of the heavy chain CDR sequences described above may also includeamino acids added to either end of the CDRs. Preparation of variants andderivatives of antibodies and antigen-binding compounds of the presentinvention, including affinity maturation or preparation of variants orderivatives containing amino acid analogs, is described in furtherdetail herein. Exemplary variants include those containing aconservative or non-conservative substitution of a corresponding aminoacid within the amino acid sequence, or a replacement of an amino acidwith a corresponding amino acid of a different human antibody sequence.

Antibodies comprising any one of the heavy chains described above mayfurther comprise a light chain, preferably a light chain that binds totarget antigen, and most preferably a light chain comprising light chainCDR sequences described below.

Another aspect of the present disclosure provides an antibody that bindstarget antigen comprising a light chain that comprises any one, two,and/or three of the light chain CDR sequences described below.

Preferably the light chain comprises an amino acid sequence identifiedas a light chain CDR3 sequence. Such a “light chain CDR3 sequence”(LCDR3) includes an amino acid sequence identified as a light chain CDR3sequence in Table 1 and within SEQ ID NOs: 18, 24 and 30. Alternatively,the light chain CDR3 sequence comprises an amino acid sequence thatcontains one or more amino acid changes (e.g., a substitution, insertionor deletion) compared to any light chain CDR3 amino acid sequenceidentified in Table 1. Preferable substitutions include a substitutionto an amino acid at the corresponding position within another lightchain CDR3 of Table 1.

The light chain comprising a light chain CDR3 sequence described abovemay further comprise a “light chain CDR1 sequence”, which includes anyof the amino acid sequences identified as a light chain CDR1 in SEQ IDNOs: 16, 22, and 28 or Table 1, amino acid sequences that contain one ormore amino acid changes compared to any light chain CDR1 identified inSEQ ID NOs: 16, 22, and 28 or Table 1, preferably a substitution to anamino acid at the corresponding position within another light chain CDR1of Table 1.

Alternatively, the light chain comprising a light chain CDR3 sequencedescribed above may further comprise a “light chain CDR2 sequence”,which includes any of the amino acid sequences identified as a lightchain CDR2 in SEQ ID NOs: 17, 23 and 29 or Table 1, amino acid sequencesthat contain one or more amino acid changes compared to any light chainCDR2 identified in Table 1, preferably a substitution to an amino acidat the corresponding position within another light chain CDR2 of SEQ IDNOs: 17, 23 and 29 or Table 1.

In a related aspect, the present disclosure contemplates a purifiedpolypeptide comprising at least one HCDR of SEQ ID NOs: 13-15, 19-21 and25-27 or LCDR of SEQ ID NOs: 16-18, 22-24 and 28-30, wherein theframework regions of the heavy chain variable region and the frameworkregions of the light chain variable region comprise framework regionsfrom a human antibody. In another embodiment, the framework regions ofthe heavy chain variable region and the framework regions of the lightchain variable region are chemically altered by amino acid substitutionto be more homologous to a different human antibody sequence. Forexample, within each heavy chain framework region (H-FR1-4) it iscontemplated that at least one, at least two, at least three, at leastfour, at least five, or at least six native framework region residues ofthe heavy chain variable region have been altered by amino acidsubstitution, and wherein within each light chain framework region(L-FR1-4), at least one, at least two, at least three, at least four, atleast five or at least six native framework residues of the light chainvariable region have been altered by amino acid substitution.

The light chain comprising a light chain CDR3 sequence described abovemay also comprise both (a) a light chain CDR1 sequence described aboveand (b) a light chain CDR2 sequence described above.

Antibodies comprising any one of the light chain variable regionsdescribed above may further comprise a heavy chain variable region,optionally paired as described in Table 1, preferably a heavy chainvariable region that binds to target antigen, and most preferably aheavy chain variable region comprising heavy chain CDR sequencesdescribed above.

In yet another embodiment, the antibody comprises a heavy chain variableregion selected from the group consisting of SEQ ID NOs: 2, 6, and 10and a light chain variable region selected from the group consisting ofSEQ ID NOs: 4, 8 and 12.

In a related aspect, the nucleic acid molecule comprises a nucleotidesequence that encodes the light chain amino acid sequence of one of SEQID NOs: 4, 8 and 12 or a portion thereof. In one embodiment, the nucleicacid molecule comprises the light chain nucleotide sequence of any oneof SEQ ID NOs: 3, 7 and 11 or a portion thereof. Nucleic acid moleculesof the disclosure further include all nucleic acid sequences, includingthe sequences in SEQ ID NOs: 1, 3, 5, 7, 9 and 11 and nucleic acidsequences comprises degenerate codons based on the diversity of thegenetic code, encoding an amino acid sequence of the heavy and lightchain variable regions of an antibody described herein or any HCDRs orLCDRs described herein, and as set out in SEQ ID NOs: 2, 4, 6, 8, 10, 12and 13-30, as well as nucleic acids that hybridize under highlystringent conditions, such as those described herein, to a nucleic acidsequence encoding an amino acid sequence of the heavy and light chainvariable regions of an antibody described herein or any HCDRs or LCDRsdescribed herein, and as set out in SEQ ID NOs: 2, 4, 6, 8, 10, 12 and13-30.

In some embodiments, the nucleic acid molecule encodes a VL amino acidsequence that is at least 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94,95, 96 97, 98 or 99% identical to a VL amino acid sequence set out inSEQ ID NOs: 4, 8 and 12. Nucleic acid molecules of the disclosureinclude nucleic acids that hybridize under highly stringent conditions,such as those described herein, to a nucleic acid sequence encoding thelight chain variable region amino acid sequence of SEQ ID NOs: 4, 8 and12, or that has the light chain variable region nucleic acid sequence ofSEQ ID NOs: 3, 7 and 11.

It is further contemplated that a nucleic acid molecule of thedisclosure comprises a nucleotide sequence that encodes the VH aminoacid sequence of any one of antibodies XPA.42.068, XPA.42.089 andXPA.42.681, or a portion thereof. In some embodiments, the nucleic acidencodes the amino acid sequence of the heavy chain CDRs of saidantibody. In some embodiments, said portion is a contiguous portioncomprising heavy chain CDR1-CDR3. In one embodiment, said portioncomprises at least one, two or three of a heavy chain CDR1, CDR2, orCDR3 region, optionally with a different human or human consensusframework, and optionally with 1, or up to 2, or up to 3 mutations inthe collective 3 CDRs.

In a related aspect, the nucleic acid molecule comprises a nucleotidesequence that encodes the heavy chain amino acid sequence of one ofheavy chain of SEQ ID NOs: 2, 6, and 10 or a portion thereof. In oneembodiment, the nucleic acid molecule comprises the heavy chainnucleotide sequence of SEQ ID NOs: 1, 5 and 9 or a portion thereof.

In some embodiments, the nucleic acid molecule encodes a VH amino acidsequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or99% identical to a VH amino acid sequence set out in SEQ ID NOs: 2, 6,and 10. In a related aspect, the VH amino acid sequence is a consensussequence. Nucleic acid molecules of the disclosure further includenucleic acids that hybridize under highly stringent conditions, such asthose described herein, to a nucleic acid sequence encoding the heavychain variable region amino acid sequence of SEQ ID NOs: 2, 6, and 10,or that has the heavy chain variable region nucleic acid sequence of anyone of SEQ ID NOs: 1, 5 and 9.

It is further contemplated that the nucleic acids of the disclosure mayencode a full-length light chain or heavy chain of an antibody selectedfrom XPA.42.068, XPA.42.089 and XPA.42.681 wherein a full-length lightchain or full-length heavy chain comprises a light chain constant regionor a heavy chain constant region, respectively, light chain constantregions optionally include unmodified or modified kappa or lambdaregions, and heavy constant regions include unmodified or modifiedconstant regions of any of the classes, such as IgG1, IgG2, IgG3, IgG4,IgM, IgA, IgD, or IgE.

In one aspect, the full length light chain antibody comprises thesequences set out in SEQ ID NOs: 4, 8 and 12. It is further contemplatedthat the nucleotide encoding the full-length light chain encodes thesequences SEQ ID NOs: 4, 8 and 12, and comprises the nucleotidessequence set forth in SEQ ID NOs: 3, 7 and 11.

In one aspect, the full length heavy chain antibody comprises thesequences in any one of SEQ ID NOs: 2, 6, and 10. It is furthercontemplated that the nucleotide encoding the full-length heavy chainencodes the sequences heavy chain of SEQ ID NOs: 2, 6, and 10 andcomprises the nucleotides sequence set forth in any one of SEQ ID NOs:1, 5 and 9.

In further embodiments, the disclosure provides an antibody that bindstransforming growth factor beta (TGFβ)1, TGFβ2 and TGFβ3 comprising alight chain variable region and/or a heavy chain variable region,wherein (a) the light chain variable region comprises at least a CDR1selected from SEQ ID NOs: 16, 22 and 28 or sequences at least 80%identical thereto, a CDR2 selected from SEQ ID NOs: 17, 23 and 29 orsequences at least 80% identical thereto, and/or a CDR3 selected fromSEQ ID NOs: 18, 24 and 30 or sequences at least 80% identical thereto;and/or wherein (b) the heavy chain variable region comprises at least aCDR1 selected from SEQ ID NOs: 13, 19 and 25 or sequences at least 80%identical thereto, a CDR2 selected from SEQ ID NOs: 14, 20 and 26 orsequences at least 80% identical thereto, and/or a CDR3 selected fromSEQ ID NOs: 15, 21 and 27 or sequences at least 80% identical thereto.

In a related embodiment, the light chain variable region comprises atleast a CDR1 selected from SEQ ID NO: 16 or sequences at least 90%identical thereto, a CDR2 selected from SEQ ID NO: 17 or sequences atleast 90% identical thereto, and a CDR3 selected from SEQ ID NO: 18 orsequences at least 90% identical thereto; and/or the heavy chainvariable region comprises at least a CDR1 selected from SEQ ID NO: 13 orsequences at least 90% identical thereto, a CDR2 selected from SEQ IDNO: 14 or sequences at least 90% identical thereto, and a CDR3 selectedfrom SEQ ID NO: 15 or sequences at least 90% identical thereto.

In another embodiment, the light chain variable region comprises atleast a CDR1 selected from SEQ ID NO: 22 or sequences at least 90%identical thereto, a CDR2 selected from SEQ ID NO: 23 or sequences atleast 90% identical thereto, and a CDR3 selected from SEQ ID NO: 24 orsequences at least 90% identical thereto; and/or the heavy chainvariable region comprises at least a CDR1 selected from SEQ ID NO: 19 orsequences at least 90% identical thereto, a CDR2 selected from SEQ IDNO: 20 or sequences at least 90% identical thereto, and a CDR3 selectedfrom SEQ ID NO: 21 or sequences at least 90% identical thereto.

In yet another embodiment, the light chain variable region comprises atleast a CDR1 selected from SEQ ID NO: 28 or sequences at least 90%identical thereto, a CDR2 selected from SEQ ID NO: 29 or sequences atleast 90% identical thereto, and a CDR3 selected from SEQ ID NO: 30 orsequences at least 90% identical thereto; and/or the heavy chainvariable region comprises at least a CDR1 selected from SEQ ID NO: 25 orsequences at least 90% identical thereto, a CDR2 selected from SEQ IDNO: 26 or sequences at least 90% identical thereto, and a CDR3 selectedfrom SEQ ID NO: 27 or sequences at least 90% identical thereto.

In exemplary embodiments, an antibody of the disclosure comprises ahuman kappa (κ) or a human lambda (λ) light chain or an amino acidsequence derived therefrom, or a human heavy chain or a sequence derivedtherefrom, or both heavy and light chains together in a single chain,dimeric, tetrameric or other form.

Monoclonal Antibodies

Monoclonal antibody refers to an antibody obtained from a population ofsubstantially homogeneous antibodies. Monoclonal antibodies aregenerally highly specific, and may be directed against a singleantigenic site, in contrast to conventional (polyclonal) antibodypreparations that typically include different antibodies directedagainst the same or different determinants (epitopes). In addition totheir specificity, the monoclonal antibodies are advantageous in thatthey are synthesized by the homogeneous culture, uncontaminated by otherimmunoglobulins with different specificities and characteristics.

Monoclonal antibodies may be made by the hybridoma method firstdescribed by Kohler et al. (Nature, 256:495-7, 1975) (Harlow & Lane;Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press:Cold Spring Harbor, N.Y. (1988); Goding, Monoclonal Antibodies:Principles and Practice, pp. 59-103 (Academic Press, 1986), or may bemade by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567).The monoclonal antibodies may also be isolated from phage antibodylibraries using the techniques described in, for example, Clackson etal., (Nature 352:624-628, 1991) and Marks et al., (J. Mol. Biol.222:581-597, 1991). Additional methods for producing monoclonalantibodies are well-known to a person of ordinary skill in the art.

Monoclonal antibodies, such as those produced by the above methods, aresuitably separated from culture medium, ascites fluid, or serum byconventional immunoglobulin purification procedures such as, forexample, protein A-Sepharose, hydrophobic interaction chromatography(HIC), ion exchange chromatography, hydroxyapatite chromatography, gelelectrophoresis, dialysis, and/or affinity chromatography.

It is further contemplated that antibodies of the present disclosure maybe used as smaller antigen binding fragments of the antibody that arewell-known in the art and described herein.

Antibody Fragments

Antibody fragments comprise a portion of an intact full length antibody,preferably an antigen binding or variable region of the intact antibody.Examples of antibody fragments include Fab, Fab′, F(ab′)2, and Fvfragments; diabodies; linear antibodies; single-chain antibody molecules(e.g., scFv); multispecific antibody fragments such as bispecfic,trispecific, etc. antibodies (e.g., diabodies, triabodies, tetrabodies);minibody; chelating recombinant antibody; tribodies or bibodies;intrabodies; nanobodies; small modular immunopharmaceuticals (SMIP),binding-domain immunoglobulin fusion proteins; camelized antibodies; VHHcontaining antibodies; and other polypeptides formed from antibodyfragments. See for example Holliger & Hudson (Nat. Biotech. 23:1126-36(2005)).

Papain digestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, monovalent fragments consisting ofthe VL, VH, CL and CH domains each with a single antigen-binding site,and a residual “Fc” fragment, whose name reflects its ability tocrystallize readily. Pepsin treatment yields a F(ab′)2 fragment, abivalent fragment comprising two Fab fragments linked by a disulfidebridge at the hinge region, that has two “Single-chain Fv” or “scFv”antibody fragments comprise the VH and VL domains of antibody, whereinthese domains are present in a single polypeptide chain. Preferably, theFv polypeptide further comprises a polypeptide linker between the VH andVL domains that enables the Fv to form the desired structure for antigenbinding, resulting in a single-chain antibody (scFv), in which a VL andVH region are paired to form a monovalent molecule via a syntheticlinker that enables them to be made as a single protein chain (Bird etal., Science 242:423-426, 1988, and Huston et al., Proc. Natl. Acad.Sci. USA 85:5879-5883, 1988). For a review of scFv see Pluckthun, in ThePharmacology of Monoclonal Antibodies, vol. 1 13, Rosenburg and Mooreeds., Springer-Verlag, New York, pp. 269-315 (1994). An Fd fragmentconsists of the VH and CH1 domains.

Additional antibody fragments include a domain antibody (dAb) fragment(Ward et al., Nature 341:544-546, 1989) which consists of a VH domain.Diabodies are bivalent antibodies in which VH and VL domains areexpressed on a single polypeptide chain, but using a linker that is tooshort to allow for pairing between the two domains on the same chain,thereby forcing the domains to pair with complementary domains ofanother chain and creating two antigen binding sites (see e.g., EP404,097; WO 93/11161; Holliger et al., Proc. Natl. Acad. Sci. USA90:6444-6448, 1993, and Poljak et al., Structure 2:1121-1123, 1994).Diabodies can be bispecific or monospecific.

Functional heavy-chain antibodies devoid of light chains are naturallyoccurring in nurse sharks (Greenberg et al., Nature 374:168-73, 1995),wobbegong sharks (Nuttall et al., Mol Immunol. 38:313-26, 2001) andCamelidae (Hamers-Casterman et al., Nature 363: 446-8, 1993; Nguyen etal., J. Mol. Biol. 275: 413, 1998), such as camels, dromedaries, alpacasand llamas. The antigen-binding site is reduced to a single domain, theVHH domain, in these animals. These antibodies form antigen-bindingregions using only heavy chain variable region, i.e., these functionalantibodies are homodimers of heavy chains only having the structure H2L2(referred to as “heavy-chain antibodies” or “HCAbs”). Camelid VHHreportedly recombines with IgG2 and IgG3 constant regions that containhinge, CH2, and CH3 domains and lack a CH1 domain (Hamers-Casterman etal., supra). For example, llama IgG1 is a conventional (H2L2) antibodyisotype in which VH recombines with a constant region that containshinge, CH1, CH2 and CH3 domains, whereas the llama IgG2 and IgG3 areheavy chain-only isotypes that lack CH1 domains and that contain nolight chains. Camelid VHH domains have been found to bind to antigenwith high affinity (Desmyter et al., J. Biol. Chem. 276:26285-90, 2001)and possess high stability in solution (Ewert et al., Biochemistry41:3628-36, 2002). Classical VH-only fragments are difficult to producein soluble form, but improvements in solubility and specific binding canbe obtained when framework residues are altered to be more VHH-like.(See, e.g., Reichman, et al., J Immunol Methods 1999, 231:25-38.)Methods for generating antibodies having camelid heavy chains aredescribed in, for example, in U.S. Patent Publication Nos. 20050136049and 20050037421.

The variable domain of an antibody heavy-chain is the smallest fullyfunctional antigen-binding fragment with a molecular mass of only 15kDa, this entity is referred to as a nanobody (Cortez-Retamozo et al.,Cancer Research 64:2853-57, 2004). A nanobody library may be generatedfrom an immunized dromedary as described in Conrath et al., (AntimicrobAgents Chemother 45: 2807-12, 2001) or using recombinant methods asdescribed in Revets et al, Expert Opin. Biol. Ther. 5(1):111-24 (2005).

Production of bispecific Fab-scFv (“bibody”) and trispecificFab-(scFv)(2) (“tribody”) are described in Schoonjans et al. (J Immunol.165:7050-57, 2000) and Willems et al. (J Chromatogr B Analyt TechnolBiomed Life Sci. 786:161-76, 2003). For bibodies or tribodies, a scFvmolecule is fused to one or both of the VL-CL (L) and VH-CH1 (Fd)chains, e.g., to produce a tribody two scFvs are fused to C-term of Fabwhile in a bibody one scFv is fused to C-term of Fab.

A “minibody” consisting of scFv fused to CH3 via a peptide linker(hingeless) or via an IgG hinge has been described in Olafsen, et al.,Protein Eng Des Sel. 17(4):315-23, 2004.

Intrabodies are single chain antibodies which demonstrate intracellularexpression and can manipulate intracellular protein function (Biocca, etal., EMBO J. 9:101-108, 1990; Colby et al., Proc Natl Acad Sci USA.101:17616-21, 2004). Intrabodies, which comprise cell signal sequenceswhich retain the antibody construct in intracellular regions, may beproduced as described in Mhashilkar et al (EMBO J 14:1542-51, 1995) andWheeler et al. (FASEB J. 17:1733-5. 2003). Transbodies arecell-permeable antibodies in which a protein transduction domain (PTD)is fused with single chain variable fragment (scFv) antibodies Heng etal., (Med Hypotheses. 64:1105-8, 2005).

Further contemplated are antibodies that are SMIPs or binding domainimmunoglobulin fusion proteins specific for target protein. Theseconstructs are single-chain polypeptides comprising antigen bindingdomains fused to immunoglobulin domains necessary to carry out antibodyeffector functions. See e.g., WO03/041600, U.S. Patent publication20030133939 and US Patent Publication 20030118592.

One or more CDRs may be incorporated into a molecule either covalentlyor noncovalently to make it an immunoadhesin. An immunoadhesin mayincorporate the CDR(s) as part of a larger polypeptide chain, maycovalently link the CDR(s) to another polypeptide chain, or mayincorporate the CDR(s) noncovalently. The CDRs permit the immunoadhesinto specifically bind to a particular antigen of interest.

Thus, a variety of compositions comprising one, two, and/or three CDRs(e.g., a single CDR alone or in tandem, 2, 3, or other multiple repeatsof the CDRs; or combinations of 2 or 3 CDRs alone or in tandem repeats;optionally, with a spacer amino acid sequence between the CDRs orrepeats) of a heavy chain variable region or a light chain variableregion of an antibody may be generated by techniques known in the art.

Multispecific Antibodies

In some embodiments, it may be desirable to generate multispecific (e.g.bispecific) anti-target antibody having binding specificities for atleast two different epitopes of the same or different molecules.Exemplary bispecific antibodies may bind to two different epitopes ofthe target molecule. Alternatively, a target-specific antibody arm maybe combined with an arm which binds to a cell surface molecule, such asa T-cell receptor molecule (e.g., CD2 or CD3), or Fc receptors for IgG(FcγR), such as FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16) so as tofocus cellular defense mechanisms to the target. Bispecific antibodiesmay also be used to localize cytotoxic agents to cells which express ortake up the target. These antibodies possess a target-binding arm and anarm which binds the cytotoxic agent (e.g., saporin, anti-interferon-60,vinca alkaloid, ricin A chain, methotrexate or radioactive isotopehapten). Bispecific antibodies can be prepared as full length antibodiesor antibody fragments (e.g., F(ab′)2 bispecific antibodies).

According to another approach for making bispecific antibodies, theinterface between a pair of antibody molecules can be engineered tomaximize the percentage of heterodimers which are recovered fromrecombinant cell culture. The preferred interface comprises at least apart of the CH3 domain of an antibody constant domain. In this method,one or more small amino acid side chains from the interface of the firstantibody molecule are replaced with larger side chains (e.g., tyrosineor tryptophan). Compensatory “cavities” of identical or similar size tothe large side chain(s) are created on the interface of the secondantibody molecule by replacing large amino acid side chains with smallerones (e.g., alanine or threonine). This provides a mechanism forincreasing the yield of the heterodimer over other unwanted end-productssuch as homodimers. See WO96/27011.

Bispecific antibodies include cross-linked or “heteroconjugate”antibodies. For example, one of the antibodies in the heteroconjugatecan be coupled to avidin, the other to biotin. Heteroconjugateantibodies may be made using any convenient cross-linking methods.Suitable cross-linking agents are well known in the art, and aredisclosed in U.S. Pat. No. 4,676,980, along with a number ofcross-linking techniques.

Techniques for generating bispecific antibodies from antibody fragmentshave also been described in the literature. For example, bispecificantibodies can be prepared using chemical linkage. Brennan et al.,(Science 229:81-83, 1985) describe a procedure wherein intact antibodiesare proteolytically cleaved to generate F(ab′)2 fragments. Thesefragments are reduced in the presence of the dithiol complexing agentsodium arsenite to stabilize vicinal dithiols and prevent intermoleculardisulfide formation. The Fab′ fragments generated are then converted tothionitrobenzoate (TNB) derivatives. One of the Fab′-TNB derivatives isthen reconverted to the Fab′-thiol by reduction with mercaptoethylamineand is mixed with an equimolar amount of the other Fab′-TNB derivativeto form the bispecific antibody. The bispecific antibodies produced canbe used as agents for the selective immobilization of enzymes. In yet afurther embodiment, Fab′-SH fragments directly recovered from E. colican be chemically coupled in vitro to form bispecific antibodies.(Shalaby et al., J. Exp. Med. 175:217-225 (1992))

Shalaby et al., J. Exp. Med. 175:217-225 (1992) describe the productionof a fully humanized bispecific antibody F(ab′)2 molecule. Each Fab′fragment was separately secreted from E. coli and subjected to directedchemical coupling in vitro to form the bispecfic antibody. Thebispecific antibody thus formed was able to bind to cells overexpressingthe HER2 receptor and normal human T cells, as well as trigger the lyticactivity of human cytotoxic lymphocytes against human breast tumortargets.

Various techniques for making and isolating bispecific antibodyfragments directly from recombinant cell culture have also beendescribed. For example, bispecific antibodies have been produced usingleucine zippers. (Kostelny et al., J. Immunol. 148:1547-1553, 1992). Theleucine zipper peptides from the Fos and Jun proteins were linked to theFab′ portions of two different antibodies by gene fusion. The antibodyhomodimers were reduced at the hinge region to form monomers and thenre-oxidized to form the antibody heterodimers. This method can also beutilized for the production of antibody homodimers. The “diabody”technology described by Hollinger et al. (Proc. Natl. Acad. Sci. USA90:6444-48, 1993) has provided an alternative mechanism for makingbispecific antibody fragments.

The fragments comprise a heavy chain variable region (VH) connected to alight-chain variable region (VL) by a linker which is too short to allowpairing between the two domains on the same chain. Accordingly, the VHand VL domains of one fragment are forced to pair with the complementaryVL and VH domains of another fragment, thereby forming twoantigen-binding sites. Another strategy for making bispecific antibodyfragments by the use of single-chain Fv (scFv) dimers has also beenreported. See Gruber et al., J. Immunol. 152: 5368 (1994).

Alternatively, the bispecific antibody may be a “linear antibody”produced as described in Zapata et al. Protein Eng. 8:1057-62 (1995).Linear antibodies comprise a pair of tandem Fd segments (VH-CH1-VH-CH1)which form a pair of antigen binding regions. Linear antibodies can bebispecific or monospecific.

In a further embodiment, the bispecific antibody may be a chelatingrecombinant antibody (CRAb). A chelating recombinant antibody recognizesadjacent and non-overlapping epitopes of the target antigen, and isflexible enough to bind to both epitopes simultaneously (Neri et al., JMol Biol. 246:367-73, 1995).

Antibodies with more than two valencies are also contemplated. Forexample, trispecific antibodies can be prepared. (Tutt et al., J.Immunol. 147:60, 1991).

Chimeric and Humanized Antibodies

Because chimeric or humanized antibodies are less immunogenic in humansthan the parental non-human (e.g., mouse) monoclonal antibodies, theycan be used for the treatment of humans with far less risk ofanaphylaxis.

Chimeric monoclonal antibodies, in which the variable Ig domains of anon-human (e.g., mouse)monoclonal antibody are fused to human constantIg domains, can be generated using standard procedures known in the art(See Morrison et al., Proc. Natl. Acad. Sci. USA 81, 6841-6855 (1984);and, Boulianne et al, Nature 312, 643-646, (1984)).

Humanized antibodies may be achieved by a variety of methods including,for example: (1) grafting the non-human complementarity determiningregions (CDRs) onto a human framework and constant region (a processreferred to in the art as humanizing through “CDR grafting”), (2)transplanting the entire non-human variable domains, but “cloaking” themwith a human-like surface by replacement of surface residues (a processreferred to in the art as “veneering”), or, alternatively, (3)substituting human amino acids at positions determined to be unlikely toadversely effect either antigen binding or protein folding, but likelyto reduce immunogenicity in a human environment (e.g., HUMANENGINEERING™). In the present disclosure, humanized antibodies willinclude both “humanized,” “veneered” and “HUMAN ENGINEERED™” antibodies.These methods are disclosed in, e.g., Jones et al., Nature 321:522 525(1986); Morrison et al., Proc. Natl. Acad. Sci., U.S.A., 81:6851-6855(1984); Morrison and Oi, Adv. Immunol., 44:65-92 (1988); Verhoeyer etal., Science 239:1534-1536 (1988); Padlan, Molec. Immun. 28:489-498(1991); Padlan, Molec. Immunol. 31:169-217 (1994); Studnicka et al. U.S.Pat. No. 5,766,886; Studnicka et al., (Protein Engineering 7: 805-814,1994; Co et al., J. Immunol. 152, 2968-2976 (1994); Riechmann, et al.,Nature 332:323-27 (1988); and Kettleborough et al., Protein Eng.4:773-783 (1991) each of which is incorporated herein by reference.

CDR grafting involves introducing one or more of the six CDRs from themouse heavy and light chain variable Ig domains into the appropriatefour framework regions of human variable Ig domains. This technique(Riechmann, et al., Nature 332:323-27 (1988)), utilizes the conservedframework regions (FR1-FR4) as a scaffold to support the CDR loops whichare the primary contacts with antigen. A disadvantage of CDR grafting,however, is that it can result in a humanized antibody that has asubstantially lower binding affinity than the original mouse antibody,because amino acids of the framework regions can contribute to antigenbinding, and because amino acids of the CDR loops can influence theassociation of the two variable Ig domains. To maintain the affinity ofthe humanized monoclonal antibody, the CDR grafting technique can beimproved by choosing human framework regions that most closely resemblethe framework regions of the original mouse antibody, and bysite-directed mutagenesis of single amino acids within the framework orCDRs aided by computer modeling of the antigen binding site (e.g., Co etal., J. Immunol. 152, 2968-2976 (1994)).

Human Antibodies from Transgenic Animals

Human antibodies to target protein can also be produced using transgenicanimals that have no endogenous immunoglobulin production and areengineered to contain human immunoglobulin loci. For example, WO98/24893 discloses transgenic animals having a human Ig locus whereinthe animals do not produce functional endogenous immunoglobulins due tothe inactivation of endogenous heavy and light chain loci. WO 91/00906also discloses transgenic non-primate mammalian hosts capable ofmounting an immune response to an immunogen, wherein the antibodies haveprimate constant and/or variable regions, and wherein the endogenousimmunoglobulin encoding loci are substituted or inactivated. WO 96/30498and U.S. Pat. No. 6,091,001 disclose the use of the Cre/Lox system tomodify the immunoglobulin locus in a mammal, such as to replace all or aportion of the constant or variable region to form a modified antibodymolecule. WO 94/02602 discloses non-human mammalian hosts havinginactivated endogenous Ig loci and functional human Ig loci. U.S. Pat.No. 5,939,598 discloses methods of making transgenic mice in which themice lack endogenous heavy chains, and express an exogenousimmunoglobulin locus comprising one or more xenogeneic constant regions.See also, U.S. Pat. Nos. 6,114,598 6,657,103 and 6,833,268.

Using a transgenic animal described above, an immune response can beproduced to a selected antigenic molecule, and antibody producing cellscan be removed from the animal and used to produce hybridomas thatsecrete human monoclonal antibodies. Immunization protocols, adjuvants,and the like are known in the art, and are used in immunization of, forexample, a transgenic mouse as described in WO 96/33735. Thispublication discloses monoclonal antibodies against a variety ofantigenic molecules including IL-6, IL-8, TNFa, human CD4, L selectin,gp39, and tetanus toxin. The monoclonal antibodies can be tested for theability to inhibit or neutralize the biological activity orphysiological effect of the corresponding protein. WO 96/33735 disclosesthat monoclonal antibodies against IL-8, derived from immune cells oftransgenic mice immunized with IL-8, blocked IL-8 induced functions ofneutrophils. Human monoclonal antibodies with specificity for theantigen used to immunize transgenic animals are also disclosed in WO96/34096 and U.S. patent application no. 20030194404; and U.S. patentapplication no. 20030031667.

Additional transgenic animals useful to make monoclonal antibodiesinclude the Medarex HuMAb-MOUSE®, described in U.S. Pat. No. 5,770,429and Fishwild, et al. (Nat. Biotechnol. 14:845-851 (1996)), whichcontains gene sequences from unrearranged human antibody genes that codefor the heavy and light chains of human antibodies. Immunization of aHuMAb-MOUSE® enables the production of fully human monoclonal antibodiesto the target protein.

Also, Ishida et al. (Cloning Stem Cells. 4:91-102 (2002)) describes theTransChromo Mouse (TCMOUSE™) which comprises megabase-sized segments ofhuman DNA and which incorporates the entire human immunoglobulin (hIg)loci. The TCMOUSE™ has a fully diverse repertoire of hIgs, including allthe subclasses of IgGs (IgG1-G4). Immunization of the TCMOUSE™ withvarious human antigens produces antibody responses comprising humanantibodies.

See also Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551 (1993);Jakobovits et al., Nature, 362:255-258 (1993); Bruggermann et al., Yearin Immunol., 7:33 (1993); and U.S. Pat. No. 5,591,669, U.S. Pat. No.5,589,369, U.S. Pat. No. 5,545,807; and U.S. Patent Publication No.20020199213. U.S. Patent Publication No. 20030092125 describes methodsfor biasing the immune response of an animal to the desired epitope.Human antibodies may also be generated by in vitro activated B cells(see U.S. Pat. Nos. 5,567,610 and 5,229,275).

Human Antibodies from Display Technology

The development of technologies for making repertoires of recombinanthuman antibody genes, and the display of the encoded antibody fragmentson the surface of filamentous bacteriophage, has provided a means formaking human antibodies directly. The antibodies produced by phagetechnology are produced as antigen binding fragments-usually Fv or Fabfragments-in bacteria and thus lack effector functions. Effectorfunctions can be introduced by one of two strategies: The fragments canbe engineered, for example, into complete antibodies for expression inmammalian cells, or into bispecific antibody fragments with a secondbinding site capable of triggering an effector function.

The present disclosure contemplates a method for producingtarget-specific antibody or antigen-binding portion thereof comprisingthe steps of synthesizing a library of human antibodies on phage,screening the library with target protein or a portion thereof,isolating phage that bind target, and obtaining the antibody from thephage. By way of example, one method for preparing the library ofantibodies for use in phage display techniques comprises the steps ofimmunizing a non-human animal comprising human immunoglobulin loci withtarget antigen or an antigenic portion thereof to create an immuneresponse, extracting antibody producing cells from the immunized animal;isolating RNA from the extracted cells, reverse transcribing the RNA toproduce cDNA, amplifying the cDNA using a primer, and inserting the cDNAinto a phage display vector such that antibodies are expressed on thephage. Recombinant target-specific antibodies of the disclosure may beobtained in this way.

In another example, antibody producing cells can be extracted fromnon-immunized animals, RNA isolated from the extracted cells and reversetranscribed to produce cDNA, which is amplified using a primer, andinserted into a phage display vector such that antibodies are expressedon the phage. Phage-display processes mimic immune selection through thedisplay of antibody repertoires on the surface of filamentousbacteriophage, and subsequent selection of phage by their binding to anantigen of choice. One such technique is described in WO 99/10494, whichdescribes the isolation of high affinity and functional agonisticantibodies for MPL and msk receptors using such an approach. Antibodiesof the disclosure can be isolated by screening of a recombinantcombinatorial antibody library, preferably a scFv phage display library,prepared using human VL and VH cDNAs prepared from mRNA derived fromhuman lymphocytes. Methodologies for preparing and screening suchlibraries are known in the art. See e.g., U.S. Pat. No. 5,969,108. Thereare commercially available kits for generating phage display libraries(e.g., the Pharmacia Recombinant Phage Antibody System, catalog no.27-9400-01; and the Stratagene SurfZAP™ phage display kit, catalog no.240612). There are also other methods and reagents that can be used ingenerating and screening antibody display libraries (see, e.g., Ladneret al. U.S. Pat. No. 5,223,409; Kang et al. PCT Publication No. WO92/18619; Dower et al. PCT Publication No. WO 91/17271; Winter et al.PCT Publication No. WO 92/20791; Markland et al. PCT Publication No. WO92/15679; Breitling et al. PCT Publication No. WO 93/01288; McCaffertyet al. PCT Publication No. WO 92/01047; Garrard et al. PCT PublicationNo. WO 92/09690; Fuchs et al. (1991) Bio/Technology 9:1370-1372; Hay etal. (1992) Hum. Antibod. Hybridomas 3:81-85; Huse et al. (1989) Science246:1275-1281; McCafferty et al., Nature (1990) 348:552-554; Griffithset al. (1993) EMBO J 12:725-734; Hawkins et al. (1992) J. Mol. Biol.226:889-896; Clackson et al. (1991) Nature 352:624-628; Gram et al.(1992) Proc. Natl. Acad. Sci. USA 89:3576-3580; Garrad et al. (1991)Bio/Technology 9:1373-1377; Hoogenboom et al. (1991) Nuc Acid Res19:4133-4137; and Barbas et al. (1991) Proc. Natl. Acad. Sci. USA88:7978-7982.

In one embodiment, to isolate human antibodies specific for the targetantigen with the desired characteristics, a human VH and VL library arescreened to select for antibody fragments having the desiredspecificity. The antibody libraries used in this method are preferablyscFv libraries prepared and screened as described herein and in the art(McCafferty et al., PCT Publication No. WO 92/01047, McCafferty et al.,(Nature 348:552-554 (1990)); and Griffiths et al., (EMBO J 12:725-734(1993)). The scFv antibody libraries preferably are screened usingtarget protein as the antigen.

Alternatively, the Fd fragment (VH-CH1) and light chain (VL-CL) ofantibodies are separately cloned by PCR and recombined randomly incombinatorial phage display libraries, which can then be selected forbinding to a particular antigen. The Fab fragments are expressed on thephage surface, i.e., physically linked to the genes that encode them.Thus, selection of Fab by antigen binding co-selects for the Fabencoding sequences, which can be amplified subsequently. Through severalrounds of antigen binding and re-amplification, a procedure termedpanning, Fab specific for the antigen are enriched and finally isolated.

In 1994, an approach for the humanization of antibodies, called “guidedselection”, was described. Guided selection utilizes the power of thephage display technique for the humanization of mouse monoclonalantibody (See Jespers, L. S., et al., Bio/Technology 12, 899-903(1994)). For this, the Fd fragment of the mouse monoclonal antibody canbe displayed in combination with a human light chain library, and theresulting hybrid Fab library may then be selected with antigen. Themouse Fd fragment thereby provides a template to guide the selection.Subsequently, the selected human light chains are combined with a humanFd fragment library. Selection of the resulting library yields entirelyhuman Fab.

A variety of procedures have been described for deriving humanantibodies from phage-display libraries (See, for example, Hoogenboom etal., J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol,222:581-597 (1991); U.S. Pat. Nos. 5,565,332 and 5,573,905; Clackson,T., and Wells, J. A., TIBTECH 12, 173-184 (1994)). In particular, invitro selection and evolution of antibodies derived from phage displaylibraries has become a powerful tool (See Burton, D. R., and Barbas III,C. F., Adv. Immunol. 57, 191-280 (1994); Winter, G., et al., Annu. Rev.Immunol. 12, 433-455 (1994); U.S. patent publication no. 20020004215 andWO 92/01047; U.S. patent publication no. 20030190317; and U.S. Pat. Nos.6,054,287 and 5,877,293.

Watkins, “Screening of Phage-Expressed Antibody Libraries by CaptureLift,” Methods in Molecular Biology, Antibody Phage Display: Methods andProtocols 178:187-193 (2002), and U.S. patent publication no.20030044772, published Mar. 6, 2003, describe methods for screeningphage-expressed antibody libraries or other binding molecules by capturelift, a method involving immobilization of the candidate bindingmolecules on a solid support.

Fv fragments are displayed on the surface of phage, by the associationof one chain expressed as a phage protein fusion (e.g., with M13 geneIII) with the complementary chain expressed as a soluble fragment. It iscontemplated that the phage may be a filamentous phage such as one ofthe class I phages: fd, M13, f1, If1, lke, ZJ/Z, Ff and one of the classII phages Xf, Pf1 and Pf3. The phage may be M13, or fd or a derivativethereof.

Once initial human VL and VH segments are selected, “mix and match”experiments, in which different pairs of the initially selected VL andVH segments are screened for target binding, are performed to selectpreferred VL/VH pair combinations. Additionally, to further improve thequality of the antibody, the VL and VH segments of the preferred VL/VHpair(s) can be randomly mutated, preferably within the any of the CDR1,CDR2 or CDR3 region of VH and/or VL, in a process analogous to the invivo somatic mutation process responsible for affinity maturation ofantibodies during a natural immune response. This in vitro affinitymaturation can be accomplished by amplifying VL and VH regions using PCRprimers complimentary to the VH CDR1, CDR2, and CDR3, or VL CDR1, CDR2,and CDR3, respectively, which primers have been “spiked” with a randommixture of the four nucleotide bases at certain positions such that theresultant PCR products encode VL and VH segments into which randommutations have been introduced into the VH and/or VL CDR3 regions. Theserandomly mutated VL and VH segments can be rescreened for binding totarget antigen.

Following screening and isolation of an target specific antibody from arecombinant immunoglobulin display library, nucleic acid encoding theselected antibody can be recovered from the display package (e.g., fromthe phage genome) and subcloned into other expression vectors bystandard recombinant DNA techniques. If desired, the nucleic acid can befurther manipulated to create other antibody forms of the disclosure, asdescribed below. To express a recombinant human antibody isolated byscreening of a combinatorial library, the DNA encoding the antibody iscloned into a recombinant expression vector and introduced into amammalian host cell, as described herein.

It is contemplated that the phage display method may be carried out in amutator strain of bacteria or host cell. A mutator strain is a host cellwhich has a genetic defect which causes DNA replicated within it to bemutated with respect to its parent DNA. Example mutator strains areNR9046mutD5 and NR9046 mut T1.

It is also contemplated that the phage display method may be carried outusing a helper phage. This is a phage which is used to infect cellscontaining a defective phage genome and which functions to complementthe defect. The defective phage genome can be a phagemid or a phage withsome function encoding gene sequences removed. Examples of helper phagesare M13K07, M13K07 gene III no. 3; and phage displaying or encoding abinding molecule fused to a capsid protein.

Antibodies are also generated via phage display screening methods usingthe hierarchical dual combinatorial approach as disclosed in WO 92/01047in which an individual colony containing either an H or L chain clone isused to infect a complete library of clones encoding the other chain (Lor H) and the resulting two-chain specific binding member is selected inaccordance with phage display techniques such as those describedtherein. This technique is also disclosed in Marks et al,(Bio/Technology, 10:779-783 (1992)).

Methods for display of peptides on the surface of yeast, microbial andmammalian cells have also been used to identify antigen specificantibodies. See, for example, U.S. Pat. Nos. 5,348,867; 5,723,287;6,699,658; Wittrup, Curr Op. Biotech. 12:395-99 (2001); Lee et al,Trends in Biotech. 21(1) 45-52 (2003); Surgeeva et al, Adv. Drug Deliv.Rev. 58: 1622-54 (2006). Antibody libraries may be attached to yeastproteins, such as agglutinin, effectively mimicking the cell surfacedisplay of antibodies by B cells in the immune system.

In addition to phage display methods, antibodies may be isolated usingin vitro display methods and microbial cell display, including ribosomedisplay and mRNA display (Amstutz et al, Curr. Op. Biotech. 12: 400-05(2001)). Selection of polypeptide using ribosome display is described inHanes et al., (Proc. Natl Acad Sci USA, 94:4937-4942 (1997)) and U.S.Pat. Nos. 5,643,768 and 5,658,754 issued to Kawasaki. Ribosome displayis also useful for rapid large scale mutational analysis of antibodies.The selective mutagenesis approach also provides a method of producingantibodies with improved activities that can be selected using ribosomaldisplay techniques.

Amino Acid Sequence Variants

It is contemplated that modified polypeptide compositions comprisingone, two, three, four, five, and/or six CDRs of an antibody aregenerated, wherein a CDR is altered to provide increased specificity oraffinity to the target molecule. Sites within antibody CDRs aretypically modified in series, e.g., by substituting first withconservative choices (e.g., hydrophobic amino acid substituted for anon-identical hydrophobic amino acid) and then with more dissimilarchoices (e.g., hydrophobic amino acid substituted for a charged aminoacid), and then deletions or insertions may be made at the target site.For example, using the conserved framework sequences surrounding theCDRs, PCR primers complementary to these consensus sequences aregenerated to amplify the antigen-specific CDR sequence located betweenthe primer regions. Techniques for cloning and expressing nucleotide andpolypeptide sequences are well-established in the art [see e.g. Sambrooket al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold SpringHarbor, N.Y. (1989)]. The amplified CDR sequences are ligated into anappropriate plasmid. The plasmid comprising one, two, three, four, fiveand/or six cloned CDRs optionally contains additional polypeptideencoding regions linked to the CDR.

Antibody substances comprising the modified CDRs are screened forbinding affinity for the original antigen. Additionally, the antibody orpolypeptide is further tested for its ability to neutralize the activityof the target antigens. For example, antibodies of the disclosure may beanalyzed as set out in the Examples to determine their ability tointerfere with the biological activity of target antigen.

Modifications may be made by conservative or non-conservative amino acidsubstitutions described in greater detail below. “Insertions” or“deletions” are preferably in the range of about 1 to 20 amino acids,more preferably 1 to 10 amino acids. The variation may be introduced bysystematically making substitutions of amino acids in an antibodypolypeptide molecule using recombinant DNA techniques and assaying theresulting recombinant variants for activity. Nucleic acid alterationscan be made at sites that differ in the nucleic acids from differentspecies (variable positions) or in highly conserved regions (constantregions). Methods for altering antibody sequences and expressingantibody polypeptide compositions useful in the disclosure are describedin greater detail below.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intra-sequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includean antibody with an N-terminal methionyl residue or the antibody(including antibody fragment) fused to an epitope tag or a salvagereceptor epitope. Other insertional variants of the antibody moleculeinclude the fusion to a polypeptide which increases the serum half-lifeof the antibody, e.g. at the N-terminus or C-terminus.

The term “epitope tagged” refers to the antibody fused to an epitopetag. The epitope tag polypeptide has enough residues to provide anepitope against which an antibody there against can be made, yet isshort enough such that it does not interfere with activity of theantibody. The epitope tag preferably is sufficiently unique so that theantibody there against does not substantially cross-react with otherepitopes. Suitable tag polypeptides generally have at least 6 amino acidresidues and usually between about 8-50 amino acid residues (preferablybetween about 9-30 residues). Examples include the flu hemagglutinin(HA) tag polypeptide and its antibody 12CA5 (Field et al., Mol. Cell.Biol. 8: 2159-2165 (1988)); the c-myc tag and the 8F9, 3C7, 6E10, G4, B7and 9E10 antibodies thereto (Evan et al., Mol. Cell. Biol. 5:3610-16(1985)); and the Herpes Simplex virus glycoprotein D (gD) tag and itsantibody (Paborsky et al., Protein Engineering 3:547-53 (1990)). Otherexemplary tags are a poly-histidine sequence, generally around sixhistidine residues, that permits isolation of a compound so labeledusing nickel chelation. Other labels and tags, such as the FLAG® tag(Eastman Kodak, Rochester, N.Y.), well known and routinely used in theart, are embraced by the disclosure.

As used herein, the term “salvage receptor binding epitope” refers to anepitope of the Fc region of an IgG molecule (e.g., IgG1, IgG2, IgG3, orIgG4) that is responsible for increasing the in vivo serum half-life ofthe IgG molecule.

Another type of variant is an amino acid substitution variant. Thesevariants have at least one amino acid residue in the antibody moleculeremoved and a different residue inserted in its place. Substitutionalmutagenesis within any of the hypervariable or CDR regions or frameworkregions is contemplated. Conservative substitutions involve replacing anamino acid with another member of its class. Non-conservativesubstitutions involve replacing a member of one of these classes with amember of another class.

Conservative amino acid substitutions are made on the basis ofsimilarity in polarity, charge, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the residues involved.For example, nonpolar (hydrophobic) amino acids include alanine (Ala,A), leucine (Leu, L), isoleucine (Ile, I), valine (Val, V), proline(Pro, P), phenylalanine (Phe, F), tryptophan (Trp, W), and methionine(Met, M); polar neutral amino acids include glycine (Gly, G), serine(Ser, S), threonine (Thr, T), cysteine (Cys, C), tyrosine (Tyr, Y),asparagine (Asn, N), and glutamine (Gln, Q); positively charged (basic)amino acids include arginine (Arg, R), lysine (Lys, K), and histidine(His, H); and negatively charged (acidic) amino acids include asparticacid (Asp, D) and glutamic acid (Glu, E).

Any cysteine residue not involved in maintaining the proper conformationof the antibody also may be substituted, generally with serine, toimprove the oxidative stability of the molecule and prevent aberrantcrosslinking. Conversely, cysteine bond(s) may be added to the antibodyto improve its stability (particularly where the antibody is an antibodyfragment such as an Fv fragment).

Affinity Maturation

Affinity maturation generally involves preparing and screening antibodyvariants that have substitutions within the CDRs of a parent antibodyand selecting variants that have one or more improved biologicalproperties such as binding affinity relative to the parent antibody. Aconvenient way for generating such substitutional variants is affinitymaturation using phage display. Briefly, several hypervariable regionsites (e.g. 6-7 sites) may be mutated to generate all possible aminosubstitutions at each site. The antibody variants thus generated aredisplayed in a monovalent fashion from filamentous phage particles asfusions to the gene III product of M13 packaged within each particle.The phage-displayed variants are then screened for their biologicalactivity (e.g. binding affinity). See e.g., WO 92/01047, WO 93/112366,WO 95/15388 and WO 93/19172.

Current antibody affinity maturation methods belong to two mutagenesiscategories: stochastic and nonstochastic. Error prone PCR, mutatorbacterial strains (Low et al., J. Mol. Biol. 260, 359-68 (1996) Irvinget al., Immunotechnology 2, 127-143 (1996)), and saturation mutagenesis(Nishimiya et al., J. Biol. Chem. 275:12813-20 (2000); Chowdhury, P. S.Methods Mol. Biol. 178, 269-85 (2002)) are typical examples ofstochastic mutagenesis methods (Rajpal et al., Proc Natl Acad Sci USA.102:8466-71 (2005)). Nonstochastic techniques often use alanine-scanningor site-directed mutagenesis to generate limited collections of specificvariants. Some methods are described in further detail below.

Affinity maturation via panning methods—Affinity maturation ofrecombinant antibodies is commonly performed through several rounds ofpanning of candidate antibodies in the presence of decreasing amounts ofantigen. Decreasing the amount of antigen per round selects theantibodies with the highest affinity to the antigen thereby yieldingantibodies of high affinity from a large pool of starting material.Affinity maturation via panning is well known in the art and isdescribed, for example, in Huls et al. (Cancer Immunol Immunother.50:163-71 (2001)). Methods of affinity maturation using phage displaytechnologies are described elsewhere herein and known in the art (seee.g., Daugherty et al., Proc Natl Acad Sci USA. 97:2029-34 (2000)).

Look-through mutagenesis—Look-through mutagenesis (LTM) (Rajpal et al.,Proc Natl Acad Sci USA. 102:8466-71 (2005)) provides a method forrapidly mapping the antibody-binding site. For LTM, nine amino acids,representative of the major side-chain chemistries provided by the 20natural amino acids, are selected to dissect the functional side-chaincontributions to binding at every position in all six CDRs of anantibody. LTM generates a positional series of single mutations within aCDR where each “wild type” residue is systematically substituted by oneof nine selected amino acids. Mutated CDRs are combined to generatecombinatorial single-chain variable fragment (scFv) libraries ofincreasing complexity and size without becoming prohibitive to thequantitative display of all variants. After positive selection, cloneswith improved binding are sequenced, and beneficial mutations aremapped.

Error-prone PCR—Error-prone PCR involves the randomization of nucleicacids between different selection rounds. The randomization occurs at alow rate by the intrinsic error rate of the polymerase used but can beenhanced by error-prone PCR (Zaccolo et al., J. Mol. Biol. 285:775-783(1999)) using a polymerase having a high intrinsic error rate duringtranscription (Hawkins et al., J Mol Biol. 226:889-96 (1992)). After themutation cycles, clones with improved affinity for the antigen areselected using routine methods in the art.

DNA Shuffling—Nucleic acid shuffling is a method for in vitro or in vivohomologous recombination of pools of shorter or smaller polynucleotidesto produce variant polynucleotides. DNA shuffling has been described inU.S. Pat. Nos. 6,605,449, 6,489,145, WO 02/092780 and Stemmer, Proc.Natl. Acad. Sci. USA, 91:10747-51 (1994). Generally, DNA shuffling iscomprised of 3 steps: fragmentation of the genes to be shuffled withDNase I, random hybridization of fragments and reassembly or filling inof the fragmented gene by PCR in the presence of DNA polymerase (sexualPCR), and amplification of reassembled product by conventional PCR.

DNA shuffling differs from error-prone PCR in that it is an inversechain reaction. In error-prone PCR, the number of polymerase start sitesand the number of molecules grows exponentially. In contrast, in nucleicacid reassembly or shuffling of random polynucleotides the number ofstart sites and the number (but not size) of the random polynucleotidesdecreases over time.

In the case of an antibody, DNA shuffling allows the free combinatorialassociation of all of the CDR1s with all of the CDR2s with all of theCDR3s, for example. It is contemplated that multiple families ofsequences can be shuffled in the same reaction. Further, shufflinggenerally conserves the relative order, such that, for example, CDR1will not be found in the position of CDR2. Rare shufflants will containa large number of the best (e.g. highest affinity) CDRs and these rareshufflants may be selected based on their superior affinity.

The template polynucleotide which may be used in DNA shuffling may beDNA or RNA. It may be of various lengths depending on the size of thegene or shorter or smaller polynucleotide to be recombined orreassembled. Preferably, the template polynucleotide is from 50 bp to 50kb. The template polynucleotide often should be double-stranded.

It is contemplated that single-stranded or double-stranded nucleic acidpolynucleotides having regions of identity to the templatepolynucleotide and regions of heterology to the template polynucleotidemay be added to the template polynucleotide, during the initial step ofgene selection. It is also contemplated that two different but relatedpolynucleotide templates can be mixed during the initial step.

Alanine scanning—Alanine scanning mutagenesis can be performed toidentify hypervariable region residues that contribute significantly toantigen binding. Cunningham and Wells, (Science 244:1081-1085 (1989)). Aresidue or group of target residues are identified (e.g., chargedresidues such as arg, asp, his, lys, and glu) and replaced by a neutralor negatively charged amino acid (most preferably alanine orpolyalanine) to affect the interaction of the amino acids with antigen.Those amino acid locations demonstrating functional sensitivity to thesubstitutions then are refined by introducing further or other variantsat, or for, the sites of substitution.

Computer-aided design—Alternatively, or in addition, it may bebeneficial to analyze a crystal structure of the antigen-antibodycomplex to identify contact points between the antibody and antigen, orto use computer software to model such contact points. Such contactresidues and neighboring residues are candidates for substitutionaccording to the techniques elaborated herein. Once such variants aregenerated, the panel of variants is subjected to screening as describedherein and antibodies with superior properties in one or more relevantassays may be selected for further development.

Alternatively, or in addition, a variety of other affinity maturationtechniques known in the art may be used, including for exampletechniques described in published patent applications WO2009/088933;WO2009/088928; WO2009/088924; as well as Clackson et al., Nature352:624-628, 1991; Marks et al., Biotechnology 10:779-783, 1992;Virnekas et al., Nucleic Acids Res. 22:5600-5607, 1994; Glaser et al.,J. Immunol. 149:3903-3913, 1992; Jackson et al., J. Immunol.154:3310-3319, 1995; Schier et al., J. Mol. Biol. 255:28-43, 1996; andYang et al., J. Mol. Biol. 254:392-403, 1995, incorporated by referenceherein in their entirety.

Altered Glycosylation

Antibody variants can also be produced that have a modifiedglycosylation pattern relative to the parent antibody, for example,deleting one or more carbohydrate moieties found in the antibody, and/oradding one or more glycosylation sites that are not present in theantibody.

Glycosylation of antibodies is typically either N-linked or O-linked.N-linked refers to the attachment of the carbohydrate moiety to the sidechain of an asparagine residue. The tripeptide sequencesasparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain. Thepresence of either of these tripeptide sequences in a polypeptidecreates a potential glycosylation site. Thus, N-linked glycosylationsites may be added to an antibody by altering the amino acid sequencesuch that it contains one or more of these tripeptide sequences.O-linked glycosylation refers to the attachment of one of the sugarsN-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, mostcommonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used. O-linked glycosylation sites may beadded to an antibody by inserting or substituting one or more serine orthreonine residues to the sequence of the original antibody.

Fc glycans influence the binding of IgG to Fc receptors and C1q, and aretherefore important for IgG effector functions. Antibody variants withmodified Fc glycans and altered effector function may be produced. Forexample, antibodies with modified terminal sugars such as sialic acids,core fucose, bisecting N-acetylglucosamine, and mannose residues mayhave altered binding to the FcγRIIIa receptor and altered ADCC activity.In a further example, antibodies with modified terminal galactoseresidues may have altered binding to C1q and altered CDC activity (Raju,Curr. Opin. Immunol. 20: 471-78 (2008).

Also contemplated are antibody molecules with absent or reducedfucosylation that exhibit improved ADCC activity. A variety of ways areknown in the art to accomplish this. For example, ADCC effector activityis mediated by binding of the antibody molecule to the FcγRIII receptor,which has been shown to be dependent on the carbohydrate structure ofthe N-linked glycosylation at the Asn-297 of the CH2 domain.Non-fucosylated antibodies bind this receptor with increased affinityand trigger FcγRIII-mediated effector functions more efficiently thannative, fucosylated antibodies. For example, recombinant production ofnon-fucosylated antibody in CHO cells in which the alpha-1,6-fucosyltransferase enzyme has been knocked out results in antibody with100-fold increased ADCC activity (Yamane-Ohnuki et al., BiotechnolBioeng. 87:614-22 (2004)). Similar effects can be accomplished throughdecreasing the activity of this or other enzymes in the fucosylationpathway, e.g., through siRNA or antisense RNA treatment, engineeringcell lines to knockout the enzyme(s), or culturing with selectiveglycosylation inhibitors (Rothman et al., Mol Immunol. 26:1113-23(1989)). Some host cell strains, e.g. Lec13 or rat hybridoma YB2/0 cellline naturally produce antibodies with lower fucosylation levels.(Shields et al., J Biol Chem. 277:26733-40 (2002); Shinkawa et al., JBiol Chem. 278:3466-73 (2003)). An increase in the level of bisectedcarbohydrate, e.g. through recombinantly producing antibody in cellsthat overexpress GnTIII enzyme, has also been determined to increaseADCC activity (Umana et al., Nat Biotechnol. 17:176-80 (1999)). It hasbeen predicted that the absence of only one of the two fucose residuesmay be sufficient to increase ADCC activity (Ferrara et al., BiotechnolBioeng. 93:851-61 (2006)).

Variants with Altered Effector Function

Other modifications of the antibody are contemplated. In one aspect, itmay be desirable to modify the antibody of the disclosure with respectto effector function, for example, to enhance the effectiveness of theantibody in treating cancer. One method for modifying effector functionteaches that cysteine residue(s) may be introduced in the Fc region,thereby allowing interchain disulfide bond formation in this region. Thehomodimeric antibody thus generated may have improved internalizationcapability and/or increased complement-mediated cell killing andantibody-dependent cellular cytotoxicity (ADCC). See Caron et al., (J.Exp Med. 176: 1191-1195 (1992)) and Shopes, B. (J. Immunol. 148:2918-2922 (1992)). Homodimeric antibodies with enhanced anti-tumoractivity may also be prepared using heterobifunctional cross-linkers asdescribed in Wolff et al., (Cancer Research 53: 2560-2565 (1993)).Alternatively, an antibody can be engineered which has dual Fc regionsand may thereby have enhanced complement lysis and ADCC capabilities.See Stevenson et al., (Anti-Cancer Drug Design 3: 219-230 (1989)). Inaddition, it has been shown that sequences within the CDR can cause anantibody to bind to MHC Class II and trigger an unwanted helper T-cellresponse. A conservative substitution can allow the antibody to retainbinding activity yet lose its ability to trigger an unwanted T-cellresponse. Also see Steplewski et al., (Proc Natl Acad Sci USA.85:4852-56 (1998)), which described chimeric antibodies wherein a murinevariable region was joined with human gamma 1, gamma 2, gamma 3, andgamma 4 constant regions.

In certain embodiments of the present disclosure, it may be desirable touse an antibody fragment, rather than an intact antibody, to increasetumor penetration, for example. In this case, it may be desirable tomodify the antibody fragment in order to increase its serum half-life,for example, adding molecules such as PEG or other water solublepolymers, including polysaccharide polymers, to antibody fragments toincrease the half-life. This may also be achieved, for example, byincorporation of a salvage receptor binding epitope into the antibodyfragment (e.g., by mutation of the appropriate region in the antibodyfragment or by incorporating the epitope into a peptide tag that is thenfused to the antibody fragment at either end or in the middle, e.g., byDNA or peptide synthesis) (see, e.g., WO96/32478).

The salvage receptor binding epitope preferably constitutes a regionwherein any one or more amino acid residues from one or two loops of aFc domain are transferred to an analogous position of the antibodyfragment. Even more preferably, three or more residues from one or twoloops of the Fc domain are transferred. Still more preferred, theepitope is taken from the CH2 domain of the Fc region (e.g., of an IgG)and transferred to the CH1, CH3, or VH region, or more than one suchregion, of the antibody. Alternatively, the epitope is taken from theCH2 domain of the Fc region and transferred to the CL region or VLregion, or both, of the antibody fragment. See also Internationalapplications WO 97/34631 and WO 96/32478 which describe Fc variants andtheir interaction with the salvage receptor.

Thus, antibodies of the present disclosure may comprise a human Fcportion, a human consensus Fc portion, or a variant thereof that retainsthe ability to interact with the Fc salvage receptor, including variantsin which cysteines involved in disulfide bonding are modified orremoved, and/or in which the a met is added at the N-terminus and/or oneor more of the N-terminal 20 amino acids are removed, and/or regionsthat interact with complement, such as the C1q binding site, areremoved, and/or the ADCC site is removed [see, e.g., Sarmay et al.,Molec. Immunol. 29:633-9 (1992)].

Previous studies mapped the binding site on human and murine IgG for FcRprimarily to the lower hinge region composed of IgG residues 233-239.Other studies proposed additional broad segments, e.g. Gly316-Lys338 forhuman Fc receptor I, Lys274-Arg301 and Tyr407-Arg416 for human Fcreceptor III, or found a few specific residues outside the lower hinge,e.g., Asn297 and Glu318 for murine IgG2b interacting with murine Fcreceptor II. The report of the 3.2-Å crystal structure of the human IgG1Fc fragment with human Fc receptor IIIA delineated IgG1 residuesLeu234-Ser239, Asp265-G1u269, Asn297-Thr299, and Ala327-Ile332 asinvolved in binding to Fc receptor IIIA. It has been suggested based oncrystal structure that in addition to the lower hinge (Leu234-Gly237),residues in IgG CH2 domain loops FG (residues 326-330) and BC (residues265-271) might play a role in binding to Fc receptor IIA. See Shields etal., (J. Biol. Chem., 276:6591-604 (2001)), incorporated by referenceherein in its entirety. Mutation of residues within Fc receptor bindingsites can result in altered effector function, such as altered ADCC orCDC activity, or altered half-life. As described above, potentialmutations include insertion, deletion or substitution of one or moreresidues, including substitution with alanine, a conservativesubstitution, a non-conservative substitution, or replacement with acorresponding amino acid residue at the same position from a differentIgG subclass (e.g. replacing an IgG1 residue with a corresponding IgG2residue at that position).

Shields et al. reported that IgG1 residues involved in binding to allhuman Fc receptors are located in the CH2 domain proximal to the hingeand fall into two categories as follows: 1) positions that may interactdirectly with all FcR include Leu234-Pro238, Ala327, and Pro329 (andpossibly Asp265); 2) positions that influence carbohydrate nature orposition include Asp265 and Asn297. The additional IgG1 residues thataffected binding to Fc receptor II are as follows: (largest effect)Arg255, Thr256, Glu258, Ser267, Asp270, Glu272, Asp280, Arg292, Ser298,and (less effect) His268, Asn276, His285, Asn286, Lys290, Gln295,Arg301, Thr307, Leu309, Asn315, Lys322, Lys326, Pro331, Ser337, Ala339,Ala378, and Lys414. A327Q, A327S, P329A, D265A and D270A reducedbinding. In addition to the residues identified above for all FcR,additional IgG1 residues that reduced binding to Fc receptor IIIA by 40%or more are as follows: Ser239, Ser267 (Gly only), His268, Glu293,Gln295, Tyr296, Arg301, Val303, Lys338, and Asp376. Variants thatimproved binding to FcRIIIA include T256A, K290A, S298A, E333A, K334A,and A339T. Lys414 showed a 40% reduction in binding for FcRIIA andFcRIIB, Arg416 a 30% reduction for FcRIIA and FcRIIIA, Gln419 a 30%reduction to FcRIIA and a 40% reduction to FcRIIB, and Lys360 a 23%improvement to FcRIIIA See also Presta et al., (Biochem. Soc. Trans.30:487-490, 2001), incorporated herein by reference in its entirety,which described several positions in the Fc region of IgG1 were foundwhich improved binding only to specific Fc gamma receptors (R) orsimultaneously improved binding to one type of Fc gamma R and reducedbinding to another type. Selected IgG1 variants with improved binding toFc gamma RIIIa were then tested in an in vitro antibody-dependentcellular cytotoxicity (ADCC) assay and showed an enhancement in ADCCwhen either peripheral blood mononuclear cells or natural killer cellswere used.

For example, U.S. Pat. No. 6,194,551, incorporated herein by referencein its entirety, describes variants with altered effector functioncontaining mutations in the human IgG Fc region, at amino acid position329, 331 or 322 (using Kabat numbering), some of which display reducedC1q binding or CDC activity. As another example, U.S. Pat. No.6,737,056, incorporated herein by reference in its entirety, describesvariants with altered effector or Fc-gamma-receptor binding containingmutations in the human IgG Fc region, at amino acid position 238, 239,248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276,278, 280, 283, 285, 286, 289, 290, 292, 294, 295, 296, 298, 301, 303,305, 307, 309, 312, 315, 320, 322, 324, 326, 327, 329, 330, 331, 333,334, 335, 337, 338, 340, 360, 373, 376, 378, 382, 388, 389, 398, 414,416, 419, 430, 434, 435, 437, 438 or 439 (using Kabat numbering), someof which display receptor binding profiles associated with reduced ADCCor CDC activity. Of these, a mutation at amino acid position 238, 265,269, 270, 327 or 329 are stated to reduce binding to FcRI, a mutation atamino acid position 238, 265, 269, 270, 292, 294, 295, 298, 303, 324,327, 329, 333, 335, 338, 373, 376, 414, 416, 419, 435, 438 or 439 arestated to reduce binding to FcRII, and a mutation at amino acid position238, 239, 248, 249, 252, 254, 265, 268, 269, 270, 272, 278, 289, 293,294, 295, 296, 301, 303, 322, 327, 329, 338, 340, 373, 376, 382, 388,389, 416, 434, 435 or 437 is stated to reduce binding to FcRIII

U.S. Pat. No. 5,624,821, incorporated by reference herein in itsentirety, reports that C1q binding activity of an murine antibody can bealtered by mutating amino acid residue 318, 320 or 322 of the heavychain and that replacing residue 297 (Asn) results in removal of lyticactivity.

U.S. Patent Publication No. 20040132101, incorporated by referenceherein in its entirety, describes variants with mutations at amino acidpositions 240, 244, 245, 247, 262, 263, 266, 299, 313, 325, 328, or 332(using Kabat numbering) or positions 234, 235, 239, 240, 241, 243, 244,245, 247, 262, 263, 264, 265, 266, 267, 269, 296, 297, 298, 299, 313,325, 327, 328, 329, 330, or 332 (using Kabat numbering), of whichmutations at positions 234, 235, 239, 240, 241, 243, 244, 245, 247, 262,263, 264, 265, 266, 267, 269, 296, 297, 298, 299, 313, 325, 327, 328,329, 330, or 332 may reduce ADCC activity or reduce binding to an Fcgamma receptor.

Chappel et al. (Proc Natl Acad Sci USA. 88:9036-40 (1991)), incorporatedherein by reference in its entirety, report that cytophilic activity ofIgG1 is an intrinsic property of its heavy chain CH2 domain. Singlepoint mutations at any of amino acid residues 234-237 of IgG1significantly lowered or abolished its activity. Substitution of all ofIgG1 residues 234-237 (LLGG) into IgG2 and IgG4 were required to restorefull binding activity. An IgG2 antibody containing the entire ELLGGPsequence (residues 233-238) was observed to be more active thanwild-type IgG1.

Isaacs et al. (J Immunol. 161:3862-9 (1998)), incorporated herein byreference in its entirety, report that mutations within a motif criticalfor Fc gammaR binding (glutamate 233 to proline, leucine/phenylalanine234 to valine, and leucine 235 to alanine) completely preventeddepletion of target cells. The mutation glutamate 318 to alanineeliminated effector function of mouse IgG2b and also reduced the potencyof human IgG4.

Armour et al. (Mol Immunol. 40:585-93 (2003)), incorporated by referenceherein in its entirety, identified IgG1 variants which react with theactivating receptor, FcgammaRIIa, at least 10-fold less efficiently thanwildtype IgG1 but whose binding to the inhibitory receptor, FcgammaRIIb,is only four-fold reduced. Mutations were made in the region of aminoacids 233-236 and/or at amino acid positions 327, 330 and 331. See alsoWO 99/58572, incorporated by reference herein in its entirety.

Xu et al. (J Biol Chem. 269:3469-74 (1994)), incorporated by referenceherein in its entirety, report that mutating IgG1 Pro331 to Ser markedlydecreased C1q binding and virtually eliminated lytic activity. Incontrast, the substitution of Pro for Ser331 in IgG4 bestowed partiallytic activity (40%) to the IgG4 Pro331 variant.

Schuurman et al. (Mol Immunol. 38:1-8 (2001)), incorporated by referenceherein in its entirety, report that mutating one of the hinge cysteinesinvolved in the inter-heavy chain bond formation, Cys226, to serineresulted in a more stable inter-heavy chain linkage. Mutating the IgG4hinge sequence Cys-Pro-Ser-Cys to the IgG1 hinge sequenceCys-Pro-Pro-Cys also markedly stabilizes the covalent interactionbetween the heavy chains.

Angal et al. (Mol Immunol. 30:105-8 (1993)), incorporated by referenceherein in its entirety, report that mutating the serine at amino acidposition 241 in IgG4 to proline (found at that position in IgG1 andIgG2) led to the production of a homogeneous antibody, as well asextending serum half-life and improving tissue distribution compared tothe original chimeric IgG4.

Covalent Modifications

Covalent modifications of the antibody are also included within thescope of this disclosure. They may be made by chemical synthesis or byenzymatic or chemical cleavage of the antibody, if applicable. Othertypes of covalent modifications of the antibody are introduced into themolecule by reacting targeted amino acid residues of the antibody withan organic derivatizing agent that is capable of reacting with selectedside chains or the N- or C-terminal residues.

Cysteinyl residues most commonly are reacted with α-haloacetates (andcorresponding amines), such as chloroacetic acid or chloroacetamide, togive carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residuesalso are derivatized by reaction with bromotrifluoroacetone,α-bromo-β-(5-imidozoyl)propionic acid, chloroacetyl phosphate,N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyldisulfide, p-chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, orchloro-7-nitrobenzo-2-oxa-1,3-diazole.

Histidyl residues are derivatized by reaction with diethylpyrocarbonateat pH 5.5-7.0 because this agent is relatively specific for the histidylside chain. Para-bromophenacyl bromide also is useful; the reaction ispreferably performed in 0.1 M sodium cacodylate at pH 6.0.

Lysinyl and amino-terminal residues are reacted with succinic or othercarboxylic acid anhydrides. Derivatization with these agents has theeffect of reversing the charge of the lysinyl residues. Other suitablereagents for derivatizing .alpha.-amino-containing residues includeimidoesters such as methyl picolinimidate, pyridoxal phosphate,pyridoxal, chloroborohydride, trinitrobenzenesulfonic acid,O-methylisourea, 2,4-pentanedione, and transaminase-catalyzed reactionwith glyoxylate.

Arginyl residues are modified by reaction with one or severalconventional reagents, among them phenylglyoxal,2,3-butanedione,1,2-cyclohexanedione, and ninhydrin. Derivatization ofarginine residues requires that the reaction be performed in alkalineconditions because of the high pKa of the guanidine functional group.Furthermore, these reagents may react with the groups of lysine as wellas the arginine epsilon-amino group.

The specific modification of tyrosyl residues may be made, withparticular interest in introducing spectral labels into tyrosyl residuesby reaction with aromatic diazonium compounds or tetranitromethane. Mostcommonly, N-acetylimidizole and tetranitromethane are used to formO-acetyl tyrosyl species and 3-nitro derivatives, respectively. Tyrosylresidues are iodinated using ¹²⁵I or ¹³¹I to prepare labeled proteinsfor use in radioimmunoas say.

Carboxyl side groups (aspartyl or glutamyl) are selectively modified byreaction with carbodiimides (R—N.dbd.C.dbd.N—R′), where R and R′ aredifferent alkyl groups, such as 1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimide or 1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide.Furthermore, aspartyl and glutamyl residues are converted to asparaginyland glutaminyl residues by reaction with ammonium ions.

Glutaminyl and asparaginyl residues are frequently deamidated to thecorresponding glutamyl and aspartyl residues, respectively. Theseresidues are deamidated under neutral or basic conditions. Thedeamidated form of these residues falls within the scope of thisdisclosure.

Other modifications include hydroxylation of proline and lysine,phosphorylation of hydroxyl groups of seryl or threonyl residues,methylation of the α-amino groups of lysine, arginine, and histidineside chains (T. E. Creighton, Proteins: Structure and MolecularProperties, W.H. Freeman & Co., San Francisco, pp. 79-86 (1983)),acetylation of the N-terminal amine, and amidation of any C-terminalcarboxyl group.

Another type of covalent modification involves chemically orenzymatically coupling glycosides to the antibody. These procedures areadvantageous in that they do not require production of the antibody in ahost cell that has glycosylation capabilities for N- or O-linkedglycosylation. Depending on the coupling mode used, the sugar(s) may beattached to (a) arginine and histidine, (b) free carboxyl groups, (c)free sulfhydryl groups such as those of cysteine, (d) free hydroxylgroups such as those of serine, threonine, or hydroxyproline, (e)aromatic residues such as those of phenylalanine, tyrosine, ortryptophan, or (f) the amide group of glutamine. These methods aredescribed in WO87/05330 and in Aplin and Wriston, (CRC Crit. Rev.Biochem., pp. 259-306 (1981)).

Removal of any carbohydrate moieties present on the antibody may beaccomplished chemically or enzymatically. Chemical deglycosylationrequires exposure of the antibody to the compoundtrifluoromethanesulfonic acid, or an equivalent compound. This treatmentresults in the cleavage of most or all sugars except the linking sugar(N-acetylglucosamine or N-acetylgalactosamine), while leaving theantibody intact. Chemical deglycosylation is described by Hakimuddin, etal., (Arch. Biochem. Biophys. 259: 52 (1987)) and by Edge et al., (Anal.Biochem. 118: 131 (1981)). Enzymatic cleavage of carbohydrate moietieson antibodies can be achieved by the use of a variety of endo- andexo-glycosidases as described by Thotakura et al., (Meth. Enzymol. 138:350 (1987)).

Another type of covalent modification of the antibody comprises linkingthe antibody to one of a variety of nonproteinaceous polymers, e.g.,polyethylene glycol, polypropylene glycol, polyoxyethylated polyols,polyoxyethylated sorbitol, polyoxyethylated glucose, polyoxyethylatedglycerol, polyoxyalkylenes, or polysaccharide polymers such as dextran.Such methods are known in the art, see, e.g. U.S. Pat. Nos. 4,640,835;4,496,689; 4,301,144; 4,670,417; 4,791,192, 4,179,337, 4,766,106,4,179,337, 4,495,285, 4,609,546 or EP 315 456.

Derivatives

As stated above, derivative refers to polypeptides chemically modifiedby such techniques as ubiquitination, labeling (e.g., with radionuclidesor various enzymes), covalent polymer attachment such as PEGylation(derivatization with polyethylene glycol) and insertion or substitutionby chemical synthesis of amino acids such as ornithine. Derivatives ofthe antibody substance of the invention, such as a bispecific antibody,are also useful as therapeutic agents and may be produced by the methodsherein.

The conjugated moiety can be incorporated in or attached to an antibodysubstance either covalently, or through ionic, van der Waals or hydrogenbonds, e.g., incorporation of radioactive nucleotides, or biotinylatednucleotides that are recognized by streptavadin.

Polyethylene glycol (PEG) may be attached to the antibody substances toprovide a longer half-life in vivo. The PEG group may be of anyconvenient molecular weight and may be linear or branched. The averagemolecular weight of the PEG will preferably range from about 2kiloDalton (“kD”) to about 100 kDa, more preferably from about 5 kDa toabout 50 kDa, most preferably from about 5 kDa to about 10 kDa. The PEGgroups will generally be attached to the antibody substances of thedisclosure via acylation or reductive alkylation through a natural orengineered reactive group on the PEG moiety (e.g., an aldehyde, amino,thiol, or ester group) to a reactive group on the antibody substance(e.g., an aldehyde, amino, or ester group). Addition of PEG moieties toantibody substances can be carried out using techniques well-known inthe art. See, e.g., International Publication No. WO 96/11953 and U.S.Pat. No. 4,179,337.

Ligation of the antibody substance with PEG usually takes place inaqueous phase and can be easily monitored by reverse phase analyticalHPLC. The PEGylated substances are purified by preparative HPLC andcharacterized by analytical HPLC, amino acid analysis and laserdesorption mass spectrometry.

Antibody Conjugates

An antibody may be administered in its “naked” or unconjugated form, ormay be conjugated directly to other therapeutic or diagnostic agents, ormay be conjugated indirectly to carrier polymers comprising such othertherapeutic or diagnostic agents. In some embodiments the antibody isconjugated to a cytotoxic agent such as a chemotherapeutic agent, adrug, a growth inhibitory agent, a toxin (e.g., an enzymatically activetoxin of bacterial, fungal, plant, or animal origin, or fragmentsthereof), or a radioactive isotope (i.e., a radioconjugate). Suitablechemotherapeutic agents include: daunomycin, doxorubicin, methotrexate,and vindesine (Rowland et al., (1986) supra). Suitable toxins include:bacterial toxins such as diphtheria toxin; plant toxins such as ricin;small molecule toxins such as geldanamycin (Mandler et al J. Natl.Cancer Inst. 92(19):1573-81 (2000); Mandler et al., Bioorg. Med. Chem.Letters 10:1025-1028 (2000); Mandler et al., Bioconjugate Chem.13.786-91 (2002)), maytansinoids (EP 1391213; Liu et al., Proc. Natl.Acad. Sci. USA 93:8618-23 (1996)), auristatins (Doronina et al., Nat.Biotech. 21: 778-84 (2003) and calicheamicin (Lode et al., Cancer Res.58:2928 (1998); Hinman et al., Cancer Res. 53:3336-3342 (1993)).

Antibodies can be detectably labeled through the use of radioisotopes,affinity labels (such as biotin, avidin, etc.), enzymatic labels (suchas horseradish peroxidase, alkaline phosphatase, etc.) fluorescent orluminescent or bioluminescent labels (such as FITC or rhodamine, etc.),paramagnetic atoms, and the like. Procedures for accomplishing suchlabeling are well known in the art; for example, see (Sternberger, L. A.et al., J. Histochem. Cytochem. 18:315 (1970); Bayer, E. A. et al.,Meth. Enzym. 62:308 (1979); Engval, E. et al., Immunol. 109:129 (1972);Goding, J. W. J. Immunol. Meth. 13:215 (1976)).

Conjugation of antibody moieties is described in U.S. Pat. No.6,306,393. General techniques are also described in Shih et al., Int. J.Cancer 41:832-839 (1988); Shih et al., Int. J. Cancer 46:1101-1106(1990); and Shih et al., U.S. Pat. No. 5,057,313. This general methodinvolves reacting an antibody component having an oxidized carbohydrateportion with a carrier polymer that has at least one free amine functionand that is loaded with a plurality of drug, toxin, chelator, boronaddends, or other therapeutic agent. This reaction results in an initialSchiff base (imine) linkage, which can be stabilized by reduction to asecondary amine to form the final conjugate.

The carrier polymer may be, for example, an aminodextran or polypeptideof at least 50 amino acid residues. Various techniques for conjugating adrug or other agent to the carrier polymer are known in the art. Apolypeptide carrier can be used instead of aminodextran, but thepolypeptide carrier should have at least 50 amino acid residues in thechain, preferably 100-5000 amino acid residues. At least some of theamino acids should be lysine residues or glutamate or aspartateresidues. The pendant amines of lysine residues and pendant carboxylatesof glutamine and aspartate are convenient for attaching a drug, toxin,immunomodulator, chelator, boron addend or other therapeutic agent.Examples of suitable polypeptide carriers include polylysine,polyglutamic acid, polyaspartic acid, co-polymers thereof, and mixedpolymers of these amino acids and others, e.g., serines, to conferdesirable solubility properties on the resultant loaded carrier andconjugate. Examples of agents to which the antibody can be conjugatedinclude any of the cytotoxic or chemotherapeutic agents describedherein.

Alternatively, conjugated antibodies can be prepared by directlyconjugating an antibody component with a therapeutic agent. The generalprocedure is analogous to the indirect method of conjugation except thata therapeutic agent is directly attached to an oxidized antibodycomponent. For example, a carbohydrate moiety of an antibody can beattached to polyethyleneglycol to extend half-life.

Alternatively, a therapeutic agent can be attached at the hinge regionof a reduced antibody component via disulfide bond formation, or using aheterobifunctional cross-linker, such as N-succinyl3-(2-pyridyldithio)proprionate (SPDP). Yu et al., Int. J. Cancer56:244(1994). General techniques for such conjugation are well-known in theart. See, for example, Wong, Chemistry Of Protein Conjugation andCross-Linking (CRC Press 1991); Upeslacis et al., “Modification ofAntibodies by Chemical Methods,” in Monoclonal Antibodies: Principlesand Applications, Birch et al. (eds.), pages 187-230 (Wiley-Liss, Inc.1995); Price, “Production and Characterization of SyntheticPeptide-Derived Antibodies,” in Monoclonal Antibodies: Production,Engineering and Clinical Application, Ritter et al. (eds.), pages 60-84(Cambridge University Press 1995). A variety of bifunctional proteincoupling agents are known in the art, such asN-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane(IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCL), active esters (such as disuccinimidyl suberate),aldehydes (such as glutareldehyde), bis-azido compounds (such as bis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene).

Antibody Fusion Proteins

Methods of making antibody fusion proteins are well known in the art.See, e.g., U.S. Pat. No. 6,306,393. Antibody fusion proteins comprisingan interleukin-2 moiety are described by Boleti et al., Ann. Oncol.6:945 (1995), Nicolet et al., Cancer Gene Ther. 2:161 (1995), Becker etal., Proc. Nat'l Acad. Sci. USA 93:7826 (1996), Hank et al., Clin.Cancer Res. 2:1951 (1996), and Hu et al., Cancer Res. 56:4998 (1996). Inaddition, Yang et al., (Hum. Antibodies Hybridomas 6:129 (1995)),describe a fusion protein that includes an F(ab′)2 fragment and a tumornecrosis factor alpha moiety. Further examples of antibody fusionproteins are described by Pastan et al, Nat. Reviews Cancer 6: 559-65(2006).

Methods of making antibody-toxin fusion proteins in which a recombinantmolecule comprises one or more antibody components and a toxin orchemotherapeutic agent also are known to those of skill in the art. Forexample, antibody-Pseudomonas exotoxin A fusion proteins have beendescribed by Chaudhary et al., Nature 339:394 (1989), Brinkmann et al.,Proc. Nat'l Acad. Sci. USA 88:8616 (1991), Batra et al., Proc. Nat'lAcad. Sci. USA 89:5867 (1992), Friedman et al., J. Immunol. 150:3054(1993), Wels et al., Int. J. Can. 60:137 (1995), Fominaya et al., J.Biol. Chem. 271:10560 (1996), Kuan et al., Biochemistry 35:2872 (1996),and Schmidt et al., Int. J. Can. 65:538 (1996). Antibody-toxin fusionproteins containing a diphtheria toxin moiety have been described byKreitman et al., Leukemia 7:553 (1993), Nicholls et al., J. Biol. Chem.268:5302 (1993), Thompson et al., J. Biol. Chem. 270:28037 (1995), andVallera et al., Blood 88:2342 (1996). Deonarain et al., Tumor Targeting1:177 (1995), have described an antibody-toxin fusion protein having anRNase moiety, while Linardou et al., Cell Biophys. 24-25:243 (1994),produced an antibody-toxin fusion protein comprising a DNase Icomponent. Gelonin was used as the toxin moiety in the antibody-toxinfusion protein of Wang et al., Abstracts of the 209th ACS NationalMeeting, Anaheim, Calif., Apr. 2-6, 1995, Part 1, BIOT005. As a furtherexample, Dohlsten et al., Proc. Nat'l Acad. Sci. USA 91:8945 (1994),reported an antibody-toxin fusion protein comprising Staphylococcalenterotoxin-A.

Illustrative of toxins which are suitably employed in the preparation ofsuch fusion proteins are ricin, abrin, ribonuclease, DNase I,Staphylococcal enterotoxin-A, pokeweed antiviral protein, gelonin,diphtherin toxin, Pseudomonas exotoxin, and Pseudomonas endotoxin. See,for example, Pastan et al., Cell 47:641 (1986), and Goldenberg, CA—ACancer Journal for Clinicians 44:43 (1994). Other suitable toxins areknown to those of skill in the art.

Antibodies of the present disclosure may also be used in ADEPT byconjugating the antibody to a prodrug-activating enzyme which converts aprodrug (e.g., a peptidyl chemotherapeutic agent, See WO81/01145) to anactive anti-cancer drug. See, for example, WO88/07378 and U.S. Pat. No.4,975,278.

The enzyme component of the immunoconjugate useful for ADEPT includesany enzyme capable of acting on a prodrug in such a way so as to covertit into its more active, cytotoxic form.

Enzymes that are useful in the present disclosure include, but are notlimited to, alkaline phosphatase useful for convertingphosphate-containing prodrugs into free drugs; arylsulfatase useful forconverting sulfate-containing prodrugs into free drugs; cytosinedeaminase useful for converting non-toxic 5-fluorocytosine into theanti-cancer drug, 5-fluorouracil; proteases, such as serratia protease,thermolysin, subtilisin, carboxypeptidases and cathepsins (such ascathepsins B and L), that are useful for converting peptide-containingprodrugs into free drugs; D-alanylcarboxypeptidases, useful forconverting prodrugs that contain D-amino acid substituents;carbohydrate-cleaving enzymes such as α-galactosidase and neuraminidaseuseful for converting glycosylated prodrugs into free drugs; β-lactamaseuseful for converting drugs derivatized with β-lactams into free drugs;and penicillin amidases, such as penicillin V amidase or penicillin Gamidase, useful for converting drugs derivatized at their aminenitrogens with phenoxyacetyl or phenylacetyl groups, respectively, intofree drugs. Alternatively, antibodies with enzymatic activity, alsoknown in the art as abzymes, can be used to convert the prodrugs of thedisclosure into free active drugs (See, e.g., Massey, Nature 328:457-458 (1987). Antibody-abzyme conjugates can be prepared as describedherein for delivery of the abzyme to a tumor cell population.

The enzymes above can be covalently bound to the antibodies bytechniques well known in the art such as the use of theheterobifunctional crosslinking reagents discussed above. Alternatively,fusion proteins comprising at least the antigen binding region of anantibody of the disclosure linked to at least a functionally activeportion of an enzyme of the disclosure can be constructed usingrecombinant DNA techniques well known in the art (See, e.g., Neubergeret al., Nature 312:604-608 (1984))

Recombinant Production of Antibodies

DNA encoding an antibody of the present disclosure may be isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of the antibodies). Usually this requires cloningthe DNA or, preferably, mRNA (i.e., cDNA) encoding the antibodies.Cloning and sequencing is carried out using standard techniques, such asfor example polymerase chain reaction (PCR), (see, e.g., Sambrook et al.(1989) Molecular Cloning: A Laboratory Guide, Vols 1-3, Cold SpringHarbor Press; Ausubel, et al. (Eds.), Protocols in Molecular Biology,John Wiley & Sons (1994)), which are incorporated herein by reference).

Nucleotide probe reactions and other nucleotide hybridization reactionsare carried out at conditions enabling the identification ofpolynucleotides which hybridize to each other under specifiedconditions. One exemplary set of conditions is as follows: stringenthybridization at 42° C. in 50% formamide, 5×SSC, 20 mM Na.PO4, pH 6.8;and washing in 1×SSC at 55° C. for 30 minutes. Formula for calculatingequivalent hybridization conditions and/or selecting other conditions toachieve a desired level of stringency are well known. It is understoodin the art that conditions of equivalent stringency can be achievedthrough variation of temperature and buffer, or salt concentration asdescribed Ausubel, et al. (Eds.), Protocols in Molecular Biology, JohnWiley & Sons (1994), pp. 6.0.3 to 6.4.10. Modifications in hybridizationconditions can be empirically determined or precisely calculated basedon the length and the percentage of guanosine/cytosine (GC) base pairingof the probe. The hybridization conditions can be calculated asdescribed in Sambrook, et al., (Eds.), Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Laboratory Press: Cold Spring Harbor, N.Y.(1989), pp. 9.47 to 9.51

As used herein, an “isolated” nucleic acid molecule or “isolated”nucleic acid sequence is a nucleic acid molecule that is either (1)identified and separated from at least one contaminant nucleic acidmolecule with which it is ordinarily associated in the natural source ofthe nucleic acid or (2) cloned, amplified, tagged, or otherwisedistinguished from background nucleic acids such that the sequence ofthe nucleic acid of interest can be determined, is considered isolated.An isolated nucleic acid molecule is other than in the form or settingin which it is found in nature. Isolated nucleic acid moleculestherefore are distinguished from the nucleic acid molecule as it existsin natural cells. However, an isolated nucleic acid molecule includes anucleic acid molecule contained in cells that ordinarily express theantibody where, for example, the nucleic acid molecule is in achromosomal location different from that of natural cells.

One source for RNA used for cloning and sequencing is a hybridomaproduced by obtaining a B cell from the transgenic mouse and fusing theB cell to an immortal cell. An advantage of using hybridomas is thatthey can be easily screened, and a hybridoma that produces a humanmonoclonal antibody of interest selected. Alternatively, RNA can beisolated from B cells (or whole spleen) of the immunized animal. Whensources other than hybridomas are used, it may be desirable to screenfor sequences encoding immunoglobulins or immunoglobulin polypeptideswith specific binding characteristics. One method for such screening isthe use of phage display technology. Phage display is described furtherherein and is also well-known in the art. See e.g., Dower et al., WO91/17271, McCafferty et al., WO 92/01047, and Caton and Koprowski,(Proc. Natl. Acad. Sci. USA, 87:6450-54 (1990)), each of which isincorporated herein by reference. In one embodiment using phage displaytechnology, cDNA from an immunized transgenic mouse (e.g., total spleencDNA) is isolated, the polymerase chain reaction is used to amplify acDNA sequences that encode a portion of an immunoglobulin polypeptide,e.g., CDR regions, and the amplified sequences are inserted into a phagevector. cDNAs encoding peptides of interest, e.g., variable regionpeptides with desired binding characteristics, are identified bystandard techniques such as panning.

Typically the sequence encoding an entire variable region of theimmunoglobulin polypeptide is determined, however, it will sometimes byadequate to sequence only a portion of a variable region, for example,the CDR-encoding portion. Typically the portion sequenced will be atleast 30 bases in length, more often based coding for at least aboutone-third or at least about one-half of the length of the variableregion will be sequenced.

Sequencing is carried out using standard techniques (see, e.g., Sambrooket al. (1989) Molecular Cloning: A Laboratory Guide, Vols 1-3, ColdSpring Harbor Press, and Sanger, F. et al. (1977) Proc. Natl. Acad. Sci.USA 74: 5463-5467, which is incorporated herein by reference). Bycomparing the sequence of the cloned nucleic acid with publishedsequences of human immunoglobulin genes and cDNAs, one of skill willreadily be able to determine, depending on the region sequenced, (i) thegermline segment usage of the immunoglobulin polypeptide (including theisotype of the heavy chain) and (ii) the sequence of the heavy and lightchain variable regions, including sequences resulting from N-regionaddition and the process of somatic mutation. One source ofimmunoglobulin gene sequence information is the National Center forBiotechnology Information, National Library of Medicine, NationalInstitutes of Health, Bethesda, Md.

Once isolated, the DNA may be placed into expression vectors, which arethen transfected into host cells such as E. coli cells, simian COScells, human embryonic kidney 293 cells (e.g., 293E cells), Chinesehamster ovary (CHO) cells, or myeloma cells that do not otherwiseproduce immunoglobulin protein, to obtain the synthesis of monoclonalantibodies in the recombinant host cells. Recombinant production ofantibodies is well known in the art.

Expression control sequences refers to DNA sequences necessary for theexpression of an operably linked coding sequence in a particular hostorganism. The control sequences that are suitable for prokaryotes, forexample, include a promoter, optionally an operator sequence, and aribosome binding site. Eukaryotic cells are known to utilize promoters,polyadenylation signals, and enhancers.

Nucleic acid is operably linked when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, operably linkedmeans that the DNA sequences being linked are contiguous, and, in thecase of a secretory leader, contiguous and in reading phase. However,enhancers do not have to be contiguous. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adaptors or linkers are used in accordancewith conventional practice.

Cell, cell line, and cell culture are often used interchangeably and allsuch designations herein include progeny. Transformants and transformedcells include the primary subject cell and cultures derived therefromwithout regard for the number of transfers. It is also understood thatall progeny may not be precisely identical in DNA content, due todeliberate or inadvertent mutations. Mutant progeny that have the samefunction or biological activity as screened for in the originallytransformed cell are included. Where distinct designations are intended,it will be clear from the context.

In an alternative embodiment, the amino acid sequence of animmunoglobulin of interest may be determined by direct proteinsequencing. Suitable encoding nucleotide sequences can be designedaccording to a universal codon table.

Amino acid sequence variants may be prepared by introducing appropriatenucleotide changes into the encoding DNA, or by peptide synthesis. Suchvariants include, for example, deletions from, and/or insertions intoand/or substitutions of, residues within the amino acid sequences of theantibodies. Any combination of deletion, insertion, and substitution ismade to arrive at the final construct, provided that the final constructpossesses the desired characteristics. The amino acid changes also mayalter post-translational processes of the molecule, such as changing thenumber or position of glycosylation sites.

Nucleic acid molecules encoding amino acid sequence variants of theantibody are prepared by a variety of methods known in the art. Thesemethods include, but are not limited to, isolation from a natural source(in the case of naturally occurring amino acid sequence variants) orpreparation by oligonucleotide-mediated (or site-directed) mutagenesis,PCR mutagenesis, and cassette mutagenesis of an earlier prepared variantor a non-variant version of the antibody.

The present disclosure also provides isolated nucleic acid encodingantibodies of the disclosure, optionally operably linked to controlsequences recognized by a host cell, vectors and host cells comprisingthe nucleic acids, and recombinant techniques for the production of theantibodies, which may comprise culturing the host cell so that thenucleic acid is expressed and, optionally, recovering the antibody fromthe host cell culture or culture medium. Various systems and methods forantibody production are reviewed by Birch & Racher (Adv. Drug Deliv.Rev. 671-685 (2006)).

For recombinant production of the antibodies, the nucleic acid encodingit is isolated and inserted into a replicable vector for further cloning(amplification of the DNA) or for expression. DNA encoding themonoclonal antibody is readily isolated and sequenced using conventionalprocedures (e.g., by using oligonucleotide probes that are capable ofbinding specifically to genes encoding the heavy and light chains of theantibody). Many vectors are available. The vector components generallyinclude, but are not limited to, one or more of the following: a signalsequence, an origin of replication, one or more selective marker genes,an enhancer element, a promoter, and a transcription terminationsequence.

(1) Signal Sequence Component

Antibodies of the present disclosure may be produced recombinantly notonly directly, but also as a fusion polypeptide with a heterologouspolypeptide, which is preferably a signal sequence or other polypeptidehaving a specific cleavage site at the N-terminus of the mature proteinor polypeptide. The signal sequence selected preferably is one that isrecognized and processed (i.e., cleaved by a signal peptidase) by thehost cell. If prokaryotic host cells do not recognize and process thenative antibody signal sequence, the signal sequence may be substitutedby a signal sequence selected, for example, from the group of thepectate lyase (e.g., pelB) alkaline phosphatase, penicillinase, 1pp, orheat-stable enterotoxin II leaders. For yeast secretion the nativesignal sequence may be substituted by, e.g., the yeast invertase leader,α factor leader (including Saccharomyces and Kluyveromyces α-factorleaders), or acid phosphatase leader, the C. albicans glucoamylaseleader, or the signal described in WO90/13646. In mammalian cellexpression, mammalian signal sequences as well as viral secretoryleaders, for example, the herpes simplex gD signal, are available.

The DNA for such precursor region is ligated in reading frame to DNAencoding the antibody.

(2) Origin of Replication Component

Both expression and cloning vectors contain a nucleic acid sequence thatenables the vector to replicate in one or more selected host cells.Generally, in cloning vectors this sequence is one that enables thevector to replicate independently of the host chromosomal DNA, andincludes origins of replication or autonomously replicating sequences.Such sequences are well known for a variety of bacteria, yeast, andviruses. The origin of replication from the plasmid pBR322 is suitablefor most Gram-negative bacteria, the 2μ plasmid origin is suitable foryeast, and various viral origins are useful for cloning vectors inmammalian cells. Generally, the origin of replication component is notneeded for mammalian expression vectors (the SV40 origin may typicallybe used only because it contains the early promoter).

(3) Selective Marker Component

Expression and cloning vectors may contain a selective gene, also termeda selectable marker. Typical selection genes encode proteins that (a)confer resistance to antibiotics or other toxins, e.g., ampicillin,neomycin, methotrexate, tetracycline, G418, geneticin, histidinol, ormycophenolic acid (b) complement auxotrophic deficiencies, or (c) supplycritical nutrients not available from complex media, e.g., the geneencoding D-alanine racemase for Bacilli.

One example of a selection scheme utilizes a drug to arrest growth of ahost cell. Those cells that are successfully transformed with aheterologous gene produce a protein conferring drug resistance and thussurvive the selection regimen. Examples of such dominant selection usethe drugs methotrexate, neomycin, histidinol, puromycin, mycophenolicacid and hygromycin.

Another example of suitable selectable markers for mammalian cells arethose that enable the identification of cells competent to take up theantibody-encoding nucleic acid, such as DHFR, thymidine kinase,metallothionein-I and -II, preferably primate metallothionein genes,adenosine deaminase, ornithine decarboxylase, etc.

For example, cells transformed with the DHFR selection gene are firstidentified by culturing all of the transformants in a culture mediumthat contains methotrexate (Mtx), a competitive antagonist of DHFR. Anappropriate host cell when wild-type DHFR is employed is the Chinesehamster ovary (CHO) cell line deficient in DHFR activity.

Alternatively, host cells (particularly wild-type hosts that containendogenous DHFR) transformed or co-transformed with DNA sequencesencoding antibody of the disclosure, wild-type DHFR protein, and anotherselectable marker such as aminoglycoside 3′-phosphotransferase (APH) canbe selected by cell growth in medium containing a selection agent forthe selectable marker such as an aminoglycosidic antibiotic, e.g.,kanamycin, neomycin, or G418. See U.S. Pat. No. 4,965,199.

A suitable selection gene for use in yeast is the trp1 gene present inthe yeast plasmid YRp7 (Stinchcomb et al., Nature, 282: 39 (1979)). Thetrp1 gene provides a selection marker for a mutant strain of yeastlacking the ability to grow in tryptophan, for example, ATCC No. 44076or PEP4-1. Jones, (Genetics 85:12 (1977)). The presence of the trp1lesion in the yeast host cell genome then provides an effectiveenvironment for detecting transformation by growth in the absence oftryptophan. Similarly, Leu2-deficient yeast strains (ATCC 20,622 or38,626) are complemented by known plasmids bearing the Leu2 gene.Ura3-deficient yeast strains are complemented by plasmids bearing theura3 gene.

In addition, vectors derived from the 1.6 μm circular plasmid pKD1 canbe used for transformation of Kluyveromyces yeasts. Alternatively, anexpression system for large-scale production of recombinant calfchymosin was reported for K. lactis Van den Berg, (Bio/Technology, 8:135(1990)). Stable multi-copy expression vectors for secretion of maturerecombinant human serum albumin by industrial strains of Kluyveromyceshave also been disclosed (Fleer et al, Bio/Technology, 9:968-975(1991)).

(4) Promoter Component

Expression and cloning vectors usually contain a promoter that isrecognized by the host organism and is operably linked to theantibody-encoding nucleic acid. Promoters suitable for use withprokaryotic hosts include the arabinose (e.g., araB) promoter phoApromoter, β-lactamase and lactose promoter systems, alkalinephosphatase, a tryptophan (trp) promoter system, and hybrid promoterssuch as the tac promoter. However, other known bacterial promoters aresuitable. Promoters for use in bacterial systems also will contain aShine-Dalgarno (S.D.) sequence operably linked to the DNA encoding theantibody of the disclosure.

Promoter sequences are known for eukaryotes. Virtually all eukaryoticgenes have an AT-rich region located approximately 25 to 30 basesupstream from the site where transcription is initiated. Anothersequence found 70 to 80 bases upstream from the start of transcriptionof many genes is a CNCAAT region where N may be any nucleotide. At the3′ end of most eukaryotic genes is an AATAAA sequence that may be thesignal for addition of the poly A tail to the 3′ end of the codingsequence. All of these sequences are suitably inserted into eukaryoticexpression vectors.

Examples of suitable promoting sequences for use with yeast hostsinclude the promoters for 3-phosphoglycerate kinase or other glycolyticenzymes, such as enolase, glyceraldehyde-3-phosphate dehydrogenase,hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoglucose isomerase, andglucokinase.

Other yeast promoters, which are inducible promoters having theadditional advantage of transcription controlled by growth conditions,are the promoter regions for alcohol dehydrogenase 2, isocytochrome C,acid phosphatase, degradative enzymes associated with nitrogenmetabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase,and enzymes responsible for maltose and galactose utilization. Suitablevectors and promoters for use in yeast expression are further describedin EP 73,657. Yeast enhancers also are advantageously used with yeastpromoters.

Antibody transcription from vectors in mammalian host cells iscontrolled, for example, by promoters obtained from the genomes ofviruses such as Abelson leukemia virus, polyoma virus, fowlpox virus,adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcomavirus, most preferably cytomegalovirus, a retrovirus, hepatitis-B virus,Simian Virus 40 (SV40), from heterologous mammalian promoters, e.g., theactin promoter or an immunoglobulin promoter, from heat-shock promoters,provided such promoters are compatible with the host cell systems.

The early and late promoters of the SV40 virus are conveniently obtainedas an SV40 restriction fragment that also contains the SV40 viral originof replication. The immediate early promoter of the humancytomegalovirus is conveniently obtained as a HindIII E restrictionfragment. A system for expressing DNA in mammalian hosts using thebovine papilloma virus as a vector is disclosed in U.S. Pat. No.4,419,446. A modification of this system is described in U.S. Pat. No.4,601,978. See also Reyes et al., Nature 297: 598-601 (1982) onexpression of human (3-interferon cDNA in mouse cells under the controlof a thymidine kinase promoter from herpes simplex virus. Alternatively,the rous sarcoma virus long terminal repeat can be used as the promoter.

(5) Enhancer Element Component

Transcription of a DNA encoding the antibody of this disclosure byhigher eukaryotes is often increased by inserting an enhancer sequenceinto the vector. Many enhancer sequences are known from mammalian genes(globin, elastase, albumin, alpha-fetoprotein, and insulin). Typically,however, one will use an enhancer from a eukaryotic cell virus. Examplesinclude the SV40 enhancer on the late side of the replication origin (bp100-270), the cytomegalovirus early promoter enhancer, the polyomaenhancer on the late side of the replication origin, and adenovirusenhancers. See also Yaniv, Nature 297:17-18 (1982) on enhancing elementsfor activation of eukaryotic promoters. The enhancer may be spliced intothe vector at a position 5′ or 3′ to the antibody-encoding sequence, butis preferably located at a site 5′ from the promoter.

(6) Transcription Termination Component

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human, or nucleated cells from other multicellularorganisms) will also contain sequences necessary for the termination oftranscription and for stabilizing the mRNA. Such sequences are commonlyavailable from the 5′ and, occasionally 3′, untranslated regions ofeukaryotic or viral DNAs or cDNAs. These regions contain nucleotidesegments transcribed as polyadenylated fragments in the untranslatedportion of the mRNA encoding antibody. One useful transcriptiontermination component is the bovine growth hormone polyadenylationregion. See WO94/11026 and the expression vector disclosed therein.Another is the mouse immunoglobulin light chain transcriptionterminator.

(7) Selection and Transformation of Host Cells

Suitable host cells for cloning or expressing the DNA in the vectorsherein are the prokaryote, yeast, or higher eukaryote cells describedabove. Suitable prokaryotes for this purpose include eubacteria, such asGram-negative or Gram-positive organisms, for example,Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter,Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium,Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacillisuch as B. subtilis and B. licheniformis (e.g., B. licheniformis 41 Pdisclosed in DD 266,710 published Apr. 12, 1989), Pseudomonas such as P.aeruginosa, and Streptomyces. One preferred E. coli cloning host is E.coli 294 (ATCC 31,446), although other strains such as E. coli B, E.coli X1776 (ATCC 31,537), and E. coli W3110 (ATCC 27,325) are suitable.These examples are illustrative rather than limiting.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts forantibody-encoding vectors. Saccharomyces cerevisiae, or common baker'syeast, is the most commonly used among lower eukaryotic hostmicroorganisms. However, a number of other genera, species, and strainsare commonly available and useful herein, such as Schizosaccharomycespombe; Kluyveromyces hosts such as, e.g., K. lactis, K. fragilis (ATCC12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K.waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans,and K. marxianus; yarrowia (EP 402,226); Pichia pastors (EP 183,070);Candida; Trichoderma reesia (EP 244,234); Neurospora crassa;Schwanniomyces such as Schwanniomyces occidentalis; and filamentousfungi such as, e.g., Neurospora, Penicillium, Tolypocladium, andAspergillus hosts such as A. nidulans and A. niger.

Suitable host cells for the expression of glycosylated antibody arederived from multicellular organisms. Examples of invertebrate cellsinclude plant and insect cells. Numerous baculoviral strains andvariants and corresponding permissive insect host cells from hosts suchas Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedesalbopictus (mosquito), Drosophila melanogaster (fruitfly), and Bombyxmori have been identified. A variety of viral strains for transfectionare publicly available, e.g., the L-1 variant of Autographa californicaNPV and the Bm-5 strain of Bombyx mori NPV, and such viruses may be usedas the virus herein according to the present disclosure, particularlyfor transfection of Spodoptera frugiperda cells.

Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato,tobacco, lemna, and other plant cells can also be utilized as hosts.

Examples of useful mammalian host cell lines are Chinese hamster ovarycells, including CHOK1 cells (ATCC CCL61), DXB-11, DG-44, and Chinesehamster ovary cells/-DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci.USA 77: 4216 (1980)); monkey kidney CV1 line transformed by SV40 (COS-7,ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subclonedfor growth in suspension culture, (Graham et al., J. Gen Virol. 36: 59,1977); baby hamster kidney cells (BHK, ATCC CCL 10); mouse sertoli cells(TM4, Mather, (Biol. Reprod. 23: 243-251, 1980); monkey kidney cells(CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCCCRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); caninekidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCCCRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (HepG2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells(Mather et al., Annals N.Y Acad. Sci. 383: 44-68 (1982)); MRC 5 cells;FS4 cells; and a human hepatoma line (Hep G2).

Host cells are transformed or transfected with the above-describedexpression or cloning vectors for antibody production and cultured inconventional nutrient media modified as appropriate for inducingpromoters, selecting transformants, or amplifying the genes encoding thedesired sequences. In addition, novel vectors and transfected cell lineswith multiple copies of transcription units separated by a selectivemarker are particularly useful and preferred for the expression ofantibodies that bind target.

(8) Culturing the Host Cells

The host cells used to produce the antibody of this disclosure may becultured in a variety of media. Commercially available media such asHam's F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma), RPMI-1640(Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) aresuitable for culturing the host cells. In addition, any of the mediadescribed in Ham et al., (Meth. Enz. 58: 44, 1979), Barnes et al., Anal.Biochem. 102: 255 (1980), U.S. Pat. Nos. 4,767,704; 4,657,866;4,927,762; 4,560,655; or 5,122,469; WO90103430; WO 87/00195; or U.S.Pat. Re. No. 30,985 may be used as culture media for the host cells. Anyof these media may be supplemented as necessary with hormones and/orother growth factors (such as insulin, transferrin, or epidermal growthfactor), salts (such as sodium chloride, calcium, magnesium, andphosphate), buffers (such as HEPES), nucleotides (such as adenosine andthymidine), antibiotics (such as gentamicin drug), trace elements(defined as inorganic compounds usually present at final concentrationsin the micromolar range), and glucose or an equivalent energy source.Any other necessary supplements may also be included at appropriateconcentrations that would be known to those skilled in the art. Theculture conditions, such as temperature, pH, and the like, are thosepreviously used with the host cell selected for expression, and will beapparent to the ordinarily skilled artisan.

(9) Purification of Antibody

When using recombinant techniques, the antibody can be producedintracellularly, in the periplasmic space, or directly secreted into themedium, including from microbial cultures. If the antibody is producedintracellularly, as a first step, the particulate debris, either hostcells or lysed fragments, is removed, for example, by centrifugation orultrafiltration. Better et al. (Science 240:1041-43, 1988; ICSU ShortReports 10:105 (1990); and Proc. Natl. Acad. Sci. USA 90:457-461 (1993)describe a procedure for isolating antibodies which are secreted to theperiplasmic space of E. coli. [See also, (Carter et al., Bio/Technology10:163-167 (1992)].

The antibody composition prepared from microbial or mammalian cells canbe purified using, for example, hydroxylapatite chromatography cation oravian exchange chromatography, and affinity chromatography, withaffinity chromatography being the preferred purification technique. Thesuitability of protein A as an affinity ligand depends on the speciesand isotype of any immunoglobulin Fc domain that is present in theantibody. Protein A can be used to purify antibodies that are based onhuman γ1, γ2, or γ4 heavy chains (Lindmark et al., J. Immunol. Meth. 62:1-13, 1983). Protein G is recommended for all mouse isotypes and forhuman γ3 (Guss et al., EMBO J. 5:15671575 (1986)). The matrix to whichthe affinity ligand is attached is most often agarose, but othermatrices are available. Mechanically stable matrices such as controlledpore glass or poly(styrenedivinyl)benzene allow for faster flow ratesand shorter processing times than can be achieved with agarose. Wherethe antibody comprises a CH 3 domain, the Bakerbond ABX™ resin (J. T.Baker, Phillipsburg, N.J.) is useful for purification. Other techniquesfor protein purification such as fractionation on an ion-exchangecolumn, ethanol precipitation, Reverse Phase HPLC, chromatography onsilica, chromatography on heparin SEPHAROSE® chromatography on an anionor cation exchange resin (such as a polyaspartic acid column),chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are alsoavailable depending on the antibody to be recovered.

Screening Methods

Effective therapeutics depend on identifying efficacious agents devoidof significant toxicity. Antibodies may be screened for binding affinityby methods known in the art. For example, gel-shift assays, Westernblots, radiolabeled competition assay, co-fractionation bychromatography, co-precipitation, cross linking, ELISA, and the like maybe used, which are described in, for example, Current Protocols inMolecular Biology (1999) John Wiley & Sons, NY, which is incorporatedherein by reference in its entirety.

In one embodiment of the present disclosure, methods of screening forantibodies which modulate the activity of a target antigen comprisecontacting test antibodies with a target polypeptide and assaying forthe presence of a complex between the antibody and the target ligand. Insuch assays, the ligand is typically labeled. After suitable incubation,free ligand is separated from that present in bound form, and the amountof free or uncomplexed label is a measure of the ability of theparticular antibody to bind to the target ligand.

In another embodiment of the present disclosure, high throughputscreening for antibody fragments or CDRs having suitable bindingaffinity to a target polypeptide is employed. Briefly, large numbers ofdifferent small peptide test compounds are synthesized on a solidsubstrate. The peptide test antibodies are contacted with a targetpolypeptide and washed. Bound polypeptides are then detected by methodswell known in the art. Purified antibodies of the disclosure can also becoated directly onto plates for use in the aforementioned drug screeningtechniques. In addition, non-neutralizing antibodies can be used tocapture the target and immobilize it on the solid support.

Methods for assessing neutralizing biological activity of TGFβ andanti-TGFβ antibodies are known in the art. See, e.g., U.S. Pat. No.7,867,496. Examples of in vitro bioassays include: (1) induction ofcolony formation of NRK cells in soft agar in the presence of EGF(Roberts et al. (1981) Proc. Natl. Acad. Sci. USA, 78:5339-5343); (2)induction of differentiation of primitive mesenchymal cells to express acartilaginous phenotype (Seyedin et al. (1985) Proc. Natl. Acad. Sci.USA, 82:2267-2271); (3) inhibition of growth of Mv1Lu mink lungepithelial cells (Danielpour et al. (1989) J. Cell. Physiol., 138:79-86)and BBC-1 monkey kidney cells (Holley et al. (1980) Proc. Natl. Acad.Sci. USA, 77:5989-5992); (4) inhibition of mitogenesis of C3H/HeJ mousethymocytes (Wrann et al. (1987) EMBO J., 6:1633-1636); (5) inhibition ofdifferentiation of rat L6 myoblast cells (Florini et al. (1986) J. Biol.Chem., 261:16509-16513); (6) measurement of fibronectin production(Wrana et al. (1992) Cell, 71:1003-1014); (7) induction of plasminogenactivator inhibitor I (PAI-1) promoter fused to a luciferase reportergene (Abe et al. (1994) Anal. Biochem., 216:276-284); (8) sandwichenzyme-linked immunosorbent assays (Danielpour et al. (1989) GrowthFactors, 2:61-71); and (9) cellular assays described in Singh et al.(2003) Bioorg. Med. Chem. Lett., 13(24):4355-4359.

In some embodiments, antibody neutralization of TGFβ1 and TGFβ2 is atleast 2-50 fold, 10-100 fold, 2-fold, 5-fold, 10-fold, 25-fold, 50-foldor 100-fold, or 20-50%, 50-100%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%,90% or 100% more potent that neutralization of TGFβ3.

Additional methods for assessing the biological activity andneutralization of TGFβ (e.g., by TGFβ antibodies) are provided in theExamples. For example, neutralization can be measured by neutralizationassays and expressed as an IC50 value. The IC50 value can be calculatedfor a given molecule by determining the concentration of molecule neededto elicit half inhibition of the maximum biological response of a secondmolecule or cell activity. The lower the IC50, the greater the potencyof the molecule to inhibit the desired protein activity. Exemplaryneutralization assays contemplated herein include, but are not limitedto, an interleukin-11 release assay and an HT-2/IL-4 cell proliferationassay. In adddtion, a TGFβ activity assay can be carried out todetermine if the antibody inhibits one TGFβ isofrm preferentially,including a pSMAD phosphorylation assay or an rhLAP binding assay. In afurther embodiment, the antibody has a lower IC50 (i.e., better binding,greater potency) for TGFβ1 and TGFβ2 compared to TGFβ3.

Combination Therapy

In one embodiment, an antibody of the present disclosure is administeredwith a second agent useful to treat a disease or disorder as describedherein. If more than one antibody effective at binding to target antigenis identified, it is contemplated that two or more antibodies todifferent epitopes of the target antigen and/or which bindpreferentially to different isoforms of TGFβ may be mixed such that thecombination of antibodies together to provide still improved efficacyagainst a condition or disorder associated with the target polypeptide.Compositions comprising one or more antibody of the invention may beadministered to persons or mammals suffering from, or predisposed tosuffer from, a condition or disorder to be treated associated with thetarget polypeptide.

Concurrent administration of two therapeutic agents does not requirethat the agents be administered at the same time or by the same route,as long as there is an overlap in the time period during which theagents are exerting their therapeutic effect. Simultaneous or sequentialadministration is contemplated, as is administration on different daysor weeks.

A second agent may be other therapeutic agents, such as cytokines,growth factors, antibodies to other target antigens, anti-inflammatoryagents, anti-coagulant agents, agent that inhibit extracellular matrixproduction, agents that will lower or reduce blood pressure, agents thatwill reduce cholesterol, triglycerides, LDL, VLDL, or lipoprotein(a) orincrease HDL, agents that will increase or decrease levels ofcholesterol-regulating proteins, anti-neoplastic drugs or molecules. Forpatients with a hyperproliferative disorder, such as cancer or a tumor,combination with second therapeutic modalities such as radiotherapy,chemotherapy, photodynamic therapy, or surgery is also contemplated.

It is contemplated the antibody of the present disclosure and the secondagent may be given simultaneously, in the same formulation. It isfurther contemplated that the agents are administered in a separateformulation and administered concurrently, with concurrently referringto agents given within 30 minutes of each other.

In another aspect, the second agent is administered prior toadministration of the antibody composition. Prior administration refersto administration of the second agent within the range of one week priorto treatment with the antibody, up to 30 minutes before administrationof the antibody. It is further contemplated that the second agent isadministered subsequent to administration of the antibody composition.Subsequent administration is meant to describe administration from 30minutes after antibody treatment up to one week after antibodyadministration.

It is further contemplated that other adjunct therapies may beadministered, where appropriate. For example, the patient may also beadministered an extracellular matrix degrading protein, surgicaltherapy, chemotherapy, a cytotoxic agent, or radiation therapy whereappropriate.

It is further contemplated that when the antibody is administered incombination with a second agent, such as for example, wherein the secondagent is a cytokine or growth factor, or a chemotherapeutic agent, theadministration also includes use of a radiotherapeutic agent orradiation therapy. The radiation therapy administered in combinationwith an antibody composition is administered as determined by thetreating physician, and at doses typically given to patients beingtreated for cancer.

A cytotoxic agent refers to a substance that inhibits or prevents thefunction of cells and/or causes destruction of cells. The term isintended to include radioactive isotopes (e.g., I131, I125, Y90 andRe186), chemotherapeutic agents, and toxins such as enzymatically activetoxins of bacterial, fungal, plant or animal origin or synthetic toxins,or fragments thereof. A non-cytotoxic agent refers to a substance thatdoes not inhibit or prevent the function of cells and/or does not causedestruction of cells. A non-cytotoxic agent may include an agent thatcan be activated to be cytotoxic. A non-cytotoxic agent may include abead, liposome, matrix or particle (see, e.g., U.S. Patent Publications2003/0028071 and 2003/0032995 which are incorporated by referenceherein). Such agents may be conjugated, coupled, linked or associatedwith an antibody according to the disclosure.

Chemotherapeutic agents contemplated for use with the antibodies of thepresent disclosure include, but are not limited to those listed in TableI:

TABLE I Alkylating agents Nitrogen mustards mechlorethaminecyclophosphamide ifosfamide melphalan chlorambucil Nitrosoureascarmustine (BCNU) lomustine (CCNU) semustine (methyl-CCNU)Ethylenimine/Methyl-melamine thriethylenemelamine (TEM) triethylenethiophosphoramide (thiotepa) hexamethylmelamine (HMM, altretamine) Alkylsulfonates busulfan Triazines dacarbazine (DTIC) Antimetabolites FolicAcid analogs methotrexate Trimetrexate Pemetrexed (Multi-targetedantifolate) Pyrimidine analogs 5-fluorouracil fluorodeoxyuridinegemcitabine cytosine arabinoside (AraC, cytarabine) 5-azacytidine2,2′-difluorodeoxy-cytidine Purine analogs 6-mercaptopurine6-thioguanine azathioprine 2′-deoxycoformycin (pentostatin)erythrohydroxynonyl-adenine (EHNA) fludarabine phosphate2-chlorodeoxyadenosine (cladribine, 2-CdA) Type I TopoisomeraseInhibitors camptothecin topotecan irinotecan Biological responsemodifiers G-CSF GM-CSF Differentiation Agents retinoic acid derivativesHormones and antagonists Adrenocorticosteroids/antagonists prednisoneand equivalents dexamethasone ainoglutethimide Progestinshydroxyprogesterone caproate medroxyprogesterone acetate megestrolacetate Estrogens diethylstilbestrol ethynyl estradiol/equivalentsAntiestrogen tamoxifen Androgens testosterone propionatefluoxymesterone/equivalents Antiandrogens flutamidegonadotropin-releasing hormone analogs leuprolide Nonsteroidalantiandrogens flutamide Natural products Antimitotic drugs Taxanespaclitaxel Vinca alkaloids vinblastine (VLB) vincristine vinorelbineTaxotere ® (docetaxel) estramustine estramustine phosphateEpipodophylotoxins etoposide teniposide Antibiotics actimomycin Ddaunomycin (rubido-mycin) doxorubicin (adria-mycin)mitoxantroneidarubicin bleomycin splicamycin (mithramycin) mitomycinCdactinomycin aphidicolin Enzymes L-asparaginase L-arginaseRadiosensitizers metronidazole misonidazole desmethylmisonidazolepimonidazole etanidazole nimorazole RSU 1069 EO9 RB 6145 SR4233nicotinamide 5-bromodeozyuridine 5-iododeoxyuridine bromodeoxycytidineMiscellaneous agents Platinium coordination complexes cisplatinCarboplatin oxaliplatin Anthracenedione mitoxantrone Substituted ureahydroxyurea Methylhydrazine derivatives N-methylhydrazine (MIH)procarbazine Adrenocortical suppressant mitotane (o,p′-DDD)ainoglutethimide Cytokines interferon (α, β, γ) interleukin-2Photosensitizers hematoporphyrin derivatives Photofrin ® benzoporphyrinderivatives Npe6 tin etioporphyrin (SnET2) pheoboride-abacteriochlorophyll-a naphthalocyanines phthalocyanines zincphthalocyanines Radiation X-ray ultraviolet light gamma radiationvisible light infrared radiation microwave radiation

It is also contemplated that the second agent is an anti-fibrotic agent.Exemplary anti-fibrotic agents include, but are not limited to, otheragents that reduce the activity of transforming growth factor-beta(TGF-β) (including but not limited to GC-1008 (Genzyme/MedImmune);lerdelimumab (CAT-152; Trabio, Cambridge Antibody); metelimumab(CAT-192,Cambridge Antibody,); LY-2157299 (Eli Lilly); ACU-HTR-028 (OpkoHealth)) including antibodies that target one or more TGF-β isoforms,inhibitors of TGF-β receptor kinases TGFBR1 (ALK5) and TGFBR2, andmodulators of post-receptor signaling pathways; chemokine receptorsignaling; endothelin receptor antagonists including inhibitors thattarget both endothelin receptor A and B and those that selectivelytarget endothelin receptor A (including but not limited to ambrisentan;avosentan; bosentan; clazosentan; darusentan; BQ-153; FR-139317,L-744453; macitentan; PD-145065; PD-156252; PD163610;PS-433540; S-0139;sitaxentan sodium; TBC-3711; zibotentan); agents that reduce theactivity of connective tissue growth factor (CTGF) (including but notlimited to FG-3019, FibroGen), and also including otherCTGF-neutralizing antibodies; matrix metalloproteinase (MMP) inhibitors(including but not limited to MMPI-12, PUP-1 and tigapotide triflutate);agents that reduce the activity of epidermal growth factor receptor(EGFR) including but not limed to erlotinib, gefitinib, BMS-690514,cetuximab, antibodies targeting EGF receptor, inhibitors of EGF receptorkinase, and modulators of post-receptor signaling pathways; agents thatreduce the activity of platelet derived growth factor (PDGF) (includingbut not limited to Imatinib mesylate (Novartis)) and also including PDGFneutralizing antibodies, antibodies targeting PDGF receptor (PDGFR),inhibitors of PDGFR kinase activity, and post-receptor signalingpathways; agents that reduce the activity of vascular endothelial growthfactor (VEGF) (including but not limited to axitinib, bevacizumab,BIBF-1120, CDP-791, CT-322, IMC-18F1, PTC-299, and ramucirumab) and alsoincluding VEGF-neutralizing antibodies, antibodies targeting the VEGFreceptor 1 (VEGFR1, Flt-1) and VEGF receptor 2 (VEGFR2, KDR), thesoluble form of VEGFR1 (sFlt) and derivatives thereof which neutralizeVEGF, and inhibitors of VEGF receptor kinase activity; inhibitors ofmultiple receptor kinases such as BIBF-1120 which inhibits receptorkinases for vascular endothelial growth factor, fibroblast growthfactor, and platelet derived growth factor; agents that interfere withintegrin function (including but not limited to STX-100 and IMGN-388)and also including integrin targeted antibodies; agents that interferewith the pro-fibrotic activities of IL-4 (including but not limited toAER-001, AMG-317, APG-201, and sIL-4Rα) and IL-13 (including but notlimited to AER-001, AMG-317, anrukinzumab, CAT-354, cintredekinbesudotox, MK-6105, QAX-576, SB-313, SL-102, and TNX-650) and alsoincluding neutralizing anti-bodies to either cytokine, antibodies thattarget IL-4 receptor or IL-13 receptor, the soluble form of IL-4receptor or derivatives thereof that is reported to bind and neutralizeboth IL-4 and IL-13, chimeric proteins including all or part of IL-13and a toxin particularly pseudomonas endotoxin, signaling though theJAK-STAT kinase pathway; agents that interfere with epithelialmesenchymal transition including inhibitors of mTor (including but notlimited to AP-23573); agents that reduce levels of copper such astetrathiomolybdate; agents that reduce oxidative stress includingN-acetyl cysteine and tetrathiomolybdate; and interferon gamma. Alsocontemplated are agents that are inhibitors of phosphodiesterase 4(PDE4) (including but not limited to Roflumilast); inhibitors ofphosphodiesterase 5 (PDE5) (including but not limited to mirodenafil,PF-4480682, sildenafil citrate, SLx-2101, tadalafil, udenafil,UK-369003, vardenafil, and zaprinast); or modifiers of the arachidonicacid pathway including cyclooxygenase and 5-lipoxegenase inhibitors(including but not limited to Zileuton). Further contemplated arecompounds that reduce tissue remodeling or fibrosis including prolylhydrolase inhibitors (including but not limited to 1016548, CG-0089,FG-2216, FG-4497, FG-5615, FG-6513, fibrostatin A (Takeda), lufironil,P-1894B, and safironil) and peroxisome proliferator-activated receptor(PPAR)-gamma agonists.(including but not limited to pioglitazone androsiglitazone).

Other specific anti-fibrotic agents contemplated include relaxin,pirfenidone, ufironil, surifonil, CAT-192, CAT-158; ambresentan, thelin;FG-3019, a CTGF antibody; anti-EGFR antibody;a EGFR kinase inhibitor;tarceva; gefitinib; PDGF antibody, PDGFR kinase inhibitor; gleevec;BIBF-1120, VEGF, FGF, and PDGF receptor inhibitor; anti-integrinantibody; IL-4 antibody; tetrathiomolybdate, a copper chelating agent;interferon-gamma; NAC, a cysteine pro-drug; hepatocyte growth factor(HGF); KGF; angiotension receptor blockers, ACE inhibitors, rennininhibitors; COX and LO inhibitors; Zileuton; monteleukast; avastin;statins; PDE5 inhibitors, such as sildenafil, udenafil, tadalafil,vardenafil, or zaprinast; rofumilast; etanercept (Enbrel); procoagulant;prostaglandins, such as PGE2, PRX-08066, a 5HT2B receptor antagonist;cintredekin besudotox, a chimeric human IL13 conjugated to a geneticallyengineered Pseudomonas exotoxin; roflumilast, a PDE4 inhibitor; FG-3019,an anti-connective tissue growth factor human monoclonal antibody;GC-1008, a TGF-β human monoclonal antibody; treprostinil, a prostacyclinanalog; interferon-α; QAX-576, a IL13 modulator; WEB 2086, aPAF-receptor antagonist; imatinib mesylate; FG-1019; Suramin; Bosentan;IFN-1b; anti-IL-4; anti-IL-13; taurine, niacin, NF-κB antisenseoligonucleotides; and nitric oxide synthase inhibitors.

Treatment of Disorders

In another embodiment, the present disclosure provides a method forinhibiting target activity by administering a target-specific antibodyto a patient in need thereof. Any of the types of antibodies describedherein may be used therapeutically. In exemplary embodiments, the targetspecific antibody is a human, chimeric or humanized antibody. In anotherexemplary embodiment, the target is human and the patient is a humanpatient. Alternatively, the patient may be a mammal that expresses atarget protein that the target specific antibody cross-reacts with. Theantibody may be administered to a non-human mammal expressing a targetprotein with which the antibody cross-reacts (i.e. a primate) forveterinary purposes or as an animal model of human disease. Such animalmodels may be useful for evaluating the therapeutic efficacy of targetspecific antibodies of the disclosure.

In one embodiment, the disclosure provides a method for treating acondition or disorder associated with TGF-β expression comprisingadministering to a subject in need thereof a therapeutically effectiveamount of an antibody or a pharmaceutical composition as describedherein.

Exemplary conditions or disorders associated with TGFβ expression thatcan be treated with an antibody substance that binds TGFβ (e.g.,antibodies of the present disclosure) include cancers, such as lungcancer, prostate cancer, breast cancer, hepatocellular cancer,esophageal cancer, colorectal cancer, pancreatic cancer, bladder cancer,kidney cancer, ovarian cancer, stomach cancer, fibrotic cancer, glioma,and melanoma, eye (e.g., ocular, optic, ophthalmic or ophthalmological)diseases, conditions or disorders, disease, conditions or disordersassociated with fibrosis, e.g., fibroproliferative diseases, conditionsor disorders, or diseases, conditions or disorders having an associatedfibrosis.

Fibroproliferative diseases, conditions or disorders or diseases havingan associated fibrosis include those that affect any organ or tissue inthe body, including, but not limited to the skin, lung, kidney, heart,brain and eye. Fibroproliferative diseases, conditions or disorders, ordiseases having an associated fibrosis include, but are not limited topulmonary fibrosis, idiopathic pulmonary fibrosis, peribronchiolarfibrosis, interstitial lung disease, chronic obstructive pulmonarydisease (COPD), small airway disease (e.g., obstructive bronchiolitis),emphysema, adult or acute respiratory distress syndrome (ARDS), acutelung injury (ALI), pulmonary fibrosis due to infectious or toxic agents,kidney fibrosis, glomerulonephritis (GN) of all etiologies, e.g.,mesangial proliferative GN, immune GN, and crescentic GN,glomerulosclerosis, tubulointerstitial injury, renal interstitialfibrosis, renal fibrosis and all causes of renal interstitial fibrosis,renal fibrosis resulting from complications of drug exposure, includingcyclosporin treatment of transplant recipients, e.g. cyclosporintreatment, HIV-associated nephropathy, transplant necropathy, diabetickidney disease (e.g., diabetic nephropathy), nephrogenic systemicfibrosis, diabetes, idiopathic retroperitoneal fibrosis, scleroderma,liver fibrosis, hepatic diseases associated with excessive scarring andprogressive sclerosis, including liver cirrhosis due to all etiologies,disorders of the biliary tree, hepatic dysfunction attributable toinfections, fibrocystic diseases, cardiovascular diseases, such ascongestive heart failure; dilated cardiomyopathy, myocarditis, vascularstenosis, cardiac fibrosis (e.g., post-infarction cardiac fibrosis),post myocardial infarction, left ventricular hypertrophy, veno-occlusivedisease, restenosis (e.g., post-angioplasty restenosis), arteriovenousgraft failure, atherosclerosis, hypertension, hypertensive heartdisease, cardiac hypertrophy, hypertrophic cardiomyopathy, heartfailure, disease of the aorta, progressive systemic sclerosis,polymyositis, systemic lupus erythematosus, dermatomyositis, fascists,Raynaud's syndrome, rheumatoid arthritis, proliferativevitreoretinopathy, vitreoretinopathy of any etiology or fibrosisassociated with ocular surgery such as treatment of glaucoma, retinalreattachment, cataract extraction, or drainage procedures of any kind,scarring in the cornea and conjunctiva, fibrosis in the cornealendothelium, alkali burn (e.g., alkali burn to the cornea),post-cataract surgery fibrosis of the lens capsule, excess scarring thetissue around the extraocular muscles in the strabismus surgery,anterior subcapsular cataract and posterior capsule opacification,anterior segment fibrotic diseases of the eye, fibrosis of the cornealstroma (e.g., associated with corneal opacification), fibrosis of thetrabecular network (e.g., associated with glaucoma), posterior segmentfibrotic diseases of the eye, fibrovascular scarring (e.g., in retinalor choroidal vasculature of the eye), retinal fibrosis, epiretinalfibrosis, retinal gliosis, subretinal fibrosis (e.g., associated withage related macular degeneration), post-retinal and glaucoma surgery,tractional retinal detachment in association with contraction of thetissue in diabetic retinopathy, Peyronie's disease, systemic sclerosis,post-spinal cord injury, osteoporosis, Camurati-Engelmann disease,Crohn's disease, scarring, Marfan syndrome, premature ovarian failure,Alzheimer's Disease and Parkinson's Disease, fibrosis due to surgicalincisions or mechanical trauma, fibrosis associated with ocular surgery,and excessive or hypertrophic scar or keloid formation in the dermisoccurring during wound healing resulting from trauma or surgical wounds.

Exemplary eye diseases (e.g., ocular, optic, ophthalmic orophthalmological diseases), conditions or disorders, include but are notlimited to, fibroproliferative disorders, fibrosis of the eye,ophthalmic fibroses, retinal dysfunction, fibrosis associated withretinal dysfunction, wet or dry macular degeneration, proliferativevitreoretinopathy, vitreoretinopathy of any etiology, fibrosisassociated with ocular surgery such as treatment of glaucoma, retinalreattachment, cataract extraction, or drainage procedures of any kind,scarring in the cornea and conjunctiva, fibrosis in the cornealendothelium, alkali burn (e.g., alkali burn to the cornea),post-cataract surgery fibrosis of the lens capsule, excess scarring inthe tissue around the extraocular muscles in the strabismus surgery,anterior subcapsular cataract and posterior capsule opacification,anterior segment fibrotic diseases of the eye, fibrosis of the cornealstroma (e.g., associated with corneal opacification), fibrosis of thetrabecular network (e.g., associated with glaucoma), posterior segmentfibrotic diseases of the eye, fibrovascular scarring (e.g., in retinalor choroidal vasculature of the eye), retinal fibrosis, epiretinalfibrosis, retinal gliosis, subretinal fibrosis (e.g., associated withage related macular degeneration), fibrosis associated with post-retinaland glaucoma surgery, tractional retinal detachment in association withcontraction of the tissue in diabetic retinopathy.

Exemplary fibroproliferative diseases, conditions or disorders of theeye, fibrosis of the eye, ocular fibrosis or ophthalmic fibrosesinclude, but are not limited to, proliferative vitreoretinopathy,vitreoretinopathy of any etiology, fibrosis associated with retinaldysfunction, fibrosis associated with wet or dry macular degeneration,fibrosis associated with ocular surgery such as treatment of glaucoma,retinal reattachment, cataract extraction, or drainage procedures of anykind, scarring in the cornea and conjunctiva, fibrosis in the cornealendothelium, fibrosis associated with alkali burn, post-cataract surgeryfibrosis of the lens capsule, excess scarring the tissue around theextraocular muscles in the strabismus surgery, anterior subcapsularcataract and posterior capsule opacification, anterior segment fibroticdiseases of the eye, fibrosis of the corneal stroma (e.g., associatedwith corneal opacification), fibrosis of the trabecular network (e.g.,associated with glaucoma), posterior segment fibrotic diseases of theeye, fibrovascular scarring (e.g., in retinal or choroidal vasculatureof the eye), retinal fibrosis, epiretinal fibrosis, retinal gliosis,subretinal fibrosis (e.g., associated with age related maculardegeneration), fibrosis associated with post-retinal and glaucomasurgery, tractional retinal detachment in association with contractionof the tissue in diabetic retinopathy.

In various embodiments, the fibroproliferative disease, condition, ordisorders of the eye is selected from the group consisting ofproliferative vitreoretinopathy, fibrosis associated with ocularsurgery, post-cataract surgery fibrosis of the lens, fibrosis of thecorneal stroma and alkali burn.

Exemplary cancers that can be treated with an antibody substanceaccording to the present invention include cancers, such as lung cancer,prostate cancer, breast cancer, hepatocellular cancer, esophagealcancer, colorectal cancer, pancreatic cancer, bladder cancer, kidneycancer, ovarian cancer, stomach cancer, fibrotic cancer, glioma andmelanoma.

It has been observed that many human tumors (deMartin et al., EMBO J.,6: 3673 (1987), Kuppner et al., Int. J. Cancer, 42: 562 (1988)) and manytumor cell lines (Derynck et al., Cancer Res., 47: 707 (1987), Robertset al., Br. J. Cancer, 57: 594 (1988)) produce TGFβ and suggests apossible mechanism for those tumors to evade normal immunologicalsurveillance.

TGFβ isoform expression in cancer is complex and variable with differentcombinations of TGFβ isoforms having different roles in particularcancers. TGFβ molecules can act both as tumor suppressors and tumorpromoters. For example, deletion or dowregulation of TGFβ signaling inanimals can result in increased breast cancer, intestinal cancer,pancreatic cancer, colon cancer and squamous cell carcinoma, indicatingthe presence of TGFβ is important to prevent or slow tumor progression(Yang et al., Trends Immunol 31:220-27, 2010). However, overexpressionof TGFβ is known to be pro-oncogenic and increased expression isdetected in many tumor types (Yang et al., supra)

Additional complexities are also disclosed in U.S. Pat. No. 7,927,593.For example, different TGFβ isoforms appear to be more relevant todifferent types of cancers. TGFβ1 and TGFβ3 may play a greater role inovarian cancer and its progression than TGFβ2; while TGFβ1 and TGFβ2expression is greater in higher grade chondrosarcoma tumors than TGFβ3.In human breast cancer, TGFβ1 and TGFβ3 are highly expressed, with TGFβ3expression correlating with overall survival, whereas patients with nodemetastasis and positive TGFβ3 expression have poor prognostic outcomes.However, in colon cancer, TGFβ1 and TGFβ2 are more highly expressed thanTGFβ3 and are present at greater circulating levels than in cancer-freeindividuals. In gliomas, TGFβ2 is important for cell migration. From therecent studies, it is not apparent which TGFβ isoforms would most usefulto inhibit in a particular cancer and to what degree.

Infiltration of immune cells into tumor sites is thought to be a commoncontributing factor to tumor growth. These immune cell infiltrates canhave a beneficial effect by helping to clear the tumor, but can also bedetrimental effect by enabling tolerance to tumor antigens. It has beenshown that TGFβ can affect levels of immune cells in tumors (see e.g.,Yang et al., Trends Immunol 31:220-27, 2010; Flavell et al., NatureImmunol 10:554-567, 2010; Nagarau et al., Expert Opin Investig Drugs19:77-91, 2010). For example, TGFβ suppresses natural killer cells thatinfiltrate tumors in order to clear tumors from the body. TGFβ alsosuppresses activity of cytotoxic T cells and CD4+ helper T cells, celltypes which assist in clearance of tumors (Yang, supra). TGFβ also playsa role in regulating dendritic cell activity, for example by inhibitingmigration into injury sites and presentation of antigen to promote animmune response. Dendritic cells are both responsive to TGFβ and secreteTGFβ. For example, dendritic cells infiltrate tumors and take up thecells, secrete TGFβ and activate regulatory T cells, which in turn canprevent tumor clearance (Flavell et al., supra). Additionally, myeloidderived suppressor cells (MDSC) are a bone marrow derived cells thatexpand during tumor progression. MDSC inhibit T cell proliferation,suppress dendritic cell maturation, and inhibit natural killer cellactivity, thereby helping cells to evade the immune response (Li et al.,J Immunol. 182:240-49, 2009). TGFβ has been demonstrated to contributeto the effects of MDSC on inhibiting natural killer cell activity (Li etal., supra; Xiang et al., Int J Cancer124:2621-33, 2009). The role ofthe various TGFβ isoforms in each of these immune processes is unclear.Selectively targeting TGFβ isoforms and inhibiting them to varyingdegrees may be instrumental in modulating the host immune response tocombat and clear the tumor.

In certain embodiments, the antibody or composition described hereinmodulates immune cells in a tumor. In some embodiments, the antibody orcomposition increases the number of natural killer (NK) cells in a tumorand/or increases cytolytic activity of NK cells. In various embodiments,the, the antibody or composition described herein decreases the numberof regulatory T cells in a tumor and/or inhibits regulatory T cellfunction. For example, in various embodiments, the antibody orcomposition described herein inhibits inhibits ability of Tregs todown-regulate an immune response or to migrate to a site of an immuneresponse.

In various embodiments, the antibody or composition described hereincytotoxic T cells in a tumor, and/or enhances CTL activity, e.g.,boosts, increases or promotes CTL activity. For example, in variousembodiments, the antibody or composition described herein increasesperform and granzyme production by CTL and increases cytolytic activityof the CTL.

In various embodiments, the antibody or composition described hereindecreases the number of monocyte-derived stem cells (MDSC) in a tumorand/or inhibits MDSC function. For example, in various embodiments, theantibody or composition described herein inhibits the ability of MDSCsto suppress an immune response, inhibits immune suppressive activity ofMDSCs, and/or inhibits the ability of MDSCs to promote expansion and/orfunction of Tregs.

In various embodiments, the, the antibody or composition describedherein decreases the number of dendritic cells (DC) in a tumor, and/orinhibits the tolerogenic function (e.g., tolerogenic effect) ofdendritic cells. For example, in various embodiments, the antibody orcomposition described herein decreases the toleragenic effect of CD8+dendritic cells.

In various embodiments, any of antibodies XPA.42.068, XPA.42.089 orXPA.42.681 or variants thereof as described herein modulate one or moreof the immune activities described above.

As stated previously, TGFβ expression has also been implicated in theonset of various tissue fibroses, such as nephrosclerosis, pulmonaryfibrosis and cirrhosis; as well as the onset of various states, such aschronic hepatitis, rheumatoid arthritis, vascular restenosis, and keloidof skin. In some exemplary embodiments, the antibodies described hereinare used to treat fibrosis or a fibrotic condition. Exemplary fibrosisor fibrotic diseases includes, but are not limited to,glomerulonephritis, adult or acute respiratory distress syndrome (ARDS),diabetes, diabetic kidney disease, liver fibrosis, kidney fibrosis, lungfibrosis, post infarction cardiac fibrosis, fibrocystic diseases,fibrotic cancer, post myocardial infarction, left ventricularhypertrophy, pulmonary fibrosis, liver cirrhosis, veno-occlusivedisease, post-spinal cord injury, post-retinal and glaucoma surgery,post-angioplasty restenosis, renal interstitial fibrosis, arteriovenousgraft failure and scarring.

In one embodiment, treatment of these disorders or conditions in ananimal in need of said treatment, comprises administering to the animalan effective amount of an antibody or a composition comprising anantibody described herein.

The conditions treatable by methods of the present disclosure preferablyoccur in mammals. Mammals include, for example, humans and otherprimates, as well as pet or companion animals such as dogs and cats,laboratory animals such as rats, mice and rabbits, and farm animals suchas horses, pigs, sheep, and cattle.

Non-therapeutic Uses

The antibodies of the present disclosure may be used as affinitypurification agents for target or in diagnostic assays for targetprotein, e.g., detecting its expression in specific cells, tissues, orserum. The antibodies may also be used for in vivo diagnostic assays.Generally, for these purposes the antibody is labeled with aradionuclide (such as ¹¹¹In, ⁹⁹Tc, ¹⁴C ¹³¹I, ¹²⁵I, ³H, ³²P or ³⁵S) sothat the antibody can be localized using immunoscintiography.

The antibodies of the present disclosure may be employed in any knownassay method, such as competitive binding assays, direct and indirectsandwich assays, such as ELISAs, and immunoprecipitation assays. Zola,Monoclonal Antibodies: A Manual of Techniques, pp.147-158 (CRC Press,Inc. 1987). The antibodies may also be used for immunohistochemistry, tolabel tissue or cell samples using methods known in the art.

The target specific antibodies can be used in a conventionalimmunoassay, including, without limitation, an ELISA, an RIA, FACS,tissue immunohistochemistry, Western blot or immunoprecipitation, whichare all techniques well-known in the art. The antibodies of thedisclosure can be used to detect target in humans and other mammals. Thepresent disclosure provides a method for detecting target in abiological sample comprising contacting a biological sample with atarget specific antibody of the disclosure and detecting the boundantibody. In one embodiment, the target specific antibody is directlylabeled with a detectable label. In another embodiment, the targetspecific antibody (the first antibody) is unlabeled and a secondantibody or other molecule that can bind the target specific antibody islabeled. As is well known to one of skill in the art, a second antibodyis chosen that is able to specifically bind the particular species andclass of the first antibody. For example, if the target specificantibody is a human IgG, then the secondary antibody could be ananti-human-IgG. Other molecules that can bind to antibodies include,without limitation, Protein A and Protein G, both of which are availablecommercially, e.g., from Pierce Chemical Co.

It is contemplated that the immunoassays disclosed above are used for anumber of purposes. For example, the target specific antibodies can beused to detect target in cells or on the surface of cells in cellculture, or secreted into the tissue culture medium. The target specificantibodies can be used to determine the amount of target on the surfaceof cells or secreted into the tissue culture medium that have beentreated with various compounds. This method can be used to identifycompounds that are useful to inhibit or activate target expression orsecretion. According to this method, one sample of cells is treated witha test compound for a period of time while another sample is leftuntreated. If the total level of target is to be measured, the cells arelysed and the total target level is measured using one of theimmunoassays described above. The total level of target in the treatedversus the untreated cells is compared to determine the effect of thetest compound.

Labels

In some embodiments, the antibody substance is labeled to facilitate itsdetection. A “label” or a “detectable moiety” is a compositiondetectable by spectroscopic, photochemical, biochemical, immunochemical,chemical, or other physical means. For example, labels suitable for usein the present disclosure include, radioactive labels (e.g., 32P),fluorophores (e.g., fluorescein), electron dense reagents, enzymes(e.g., as commonly used in an ELISA), biotin, digoxigenin, or haptens aswell as proteins which can be made detectable, e.g., by incorporating aradiolabel into the hapten or peptide, or used to detect antibodiesspecifically reactive with the hapten or peptide.

Examples of labels suitable for use in the present invention include,but are not limited to, fluorescent dyes (e.g., fluoresceinisothiocyanate, Texas red, rhodamine, and the like), radiolabels (e.g.,³H, ¹²⁵I, ³⁵S, ¹⁴C, or ³²P), enzymes (e.g., horse radish peroxidase,alkaline phosphatase and others commonly used in an ELISA), andcolorimetric labels such as colloidal gold, colored glass or plasticbeads (e.g., polystyrene, polypropylene, latex, etc.).

The label may be coupled directly or indirectly to the desired componentof the assay according to methods well known in the art. Preferably, thelabel in one embodiment is covalently bound to the biopolymer using anisocyanate reagent for conjugation of an active agent according to thedisclosure. In one aspect of the present disclosure, the bifunctionalisocyanate reagents of the disclosure can be used to conjugate a labelto a biopolymer to form a label biopolymer conjugate without an activeagent attached thereto. The label biopolymer conjugate may be used as anintermediate for the synthesis of a labeled conjugate according to thedisclosure or may be used to detect the biopolymer conjugate. Asindicated above, a wide variety of labels can be used, with the choiceof label depending on sensitivity required, ease of conjugation with thedesired component of the assay, stability requirements, availableinstrumentation, and disposal provisions. Non-radioactive labels areoften attached by indirect means. Generally, a ligand molecule (e.g.,biotin) is covalently bound to the molecule. The ligand then binds toanother molecules (e.g., streptavidin) molecule, which is eitherinherently detectable or covalently bound to a signal system, such as adetectable enzyme, a fluorescent compound, or a chemiluminescentcompound.

The compounds of the present disclosure can also be conjugated directlyto signal-generating compounds, e.g., by conjugation with an enzyme orfluorophore. Enzymes suitable for use as labels include, but are notlimited to, hydrolases, particularly phosphatases, esterases andglycosidases, or oxidotases, particularly peroxidases. Fluorescentcompounds, i.e., fluorophores, suitable for use as labels include, butare not limited to, fluorescein and its derivatives, rhodamine and itsderivatives, dansyl, umbelliferone, etc. Further examples of suitablefluorophores include, but are not limited to, eosin, TRITC-amine,quinine, fluorescein W, acridine yellow, lissamine rhodamine, B sulfonylchloride erythroscein, ruthenium (tris, bipyridinium), Texas Red,nicotinamide adenine dinucleotide, flavin adenine dinucleotide, etc.Chemiluminescent compounds suitable for use as labels include, but arenot limited to, luciferin and 2,3-dihydrophthalazinediones, e.g.,luminol. For a review of various labeling or signal producing systemsthat can be used in the methods of the present disclosure, see U.S. Pat.No. 4,391,904.

Means for detecting labels are well known to those of skill in the art.Thus, for example, where the label is radioactive, means for detectioninclude a scintillation counter or photographic film, as inautoradiography. Where the label is a fluorescent label, it may bedetected by exciting the fluorochrome with the appropriate wavelength oflight and detecting the resulting fluorescence. The fluorescence may bedetected visually, by the use of electronic detectors such as chargecoupled devices (CCDs) or photomultipliers and the like. Similarly,enzymatic labels may be detected by providing the appropriate substratesfor the enzyme and detecting the resulting reaction product.Colorimetric or chemiluminescent labels may be detected simply byobserving the color associated with the label. Other labeling anddetection systems suitable for use in the methods of the presentdisclosure will be readily apparent to those of skill in the art. Suchlabeled modulators and ligands can be used in the diagnosis of a diseaseor health condition.

Formulation of Pharmaceutical Compositions

To administer antibody substances of the present disclosure to human ortest animals, it is preferable to formulate the antibody substances in acomposition comprising one or more pharmaceutically acceptable carriers.The phrase “pharmaceutically or pharmacologically acceptable” refer tomolecular entities and compositions that do not produce allergic, orother adverse reactions when administered using routes well-known in theart, as described below. “Pharmaceutically acceptable carriers” includeany and all clinically useful solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents and the like.

In addition, compounds may form solvates with water or common organicsolvents. Such solvates are contemplated as well.

The antibody is administered by any suitable means, includingparenteral, subcutaneous, intraperitoneal, intrapulmonary, andintranasal, and, if desired for local treatment, intralesionaladministration. Parenteral infusions include intravenous, intraarterial,intraperitoneal, intramuscular, intradermal or subcutaneousadministration. In addition, the antibody is suitably administered bypulse infusion, particularly with declining doses of the antibody.Preferably the dosing is given by injections, most preferablyintravenous or subcutaneous injections, depending in part on whether theadministration is brief or chronic. Other administration methods arecontemplated, including topical, particularly transdermal, transmucosal,rectal, oral or local administration e.g. through a catheter placedclose to the desired site. Injection, especially intravenous, ispreferred.

Pharmaceutical compositions of the present disclosure containing anantibody substance of the disclosure as an active ingredient may containpharmaceutically acceptable carriers or additives depending on the routeof administration. Examples of such carriers or additives include water,a pharmaceutical acceptable organic solvent, collagen, polyvinylalcohol, polyvinylpyrrolidone, a carboxyvinyl polymer,carboxymethylcellulose sodium, polyacrylic sodium, sodium alginate,water-soluble dextran, carboxymethyl starch sodium, pectin, methylcellulose, ethyl cellulose, xanthan gum, gum Arabic, casein, gelatin,agar, diglycerin, glycerin, propylene glycol, polyethylene glycol,Vaseline, paraffin, stearyl alcohol, stearic acid, human serum albumin(HSA), mannitol, sorbitol, lactose, a pharmaceutically acceptablesurfactant and the like. Additives used are chosen from, but not limitedto, the above or combinations thereof, as appropriate, depending on thedosage form of the present disclosure.

Formulation of the pharmaceutical composition will vary according to theroute of administration selected (e.g., solution, emulsion). Anappropriate composition comprising the antibody to be administered canbe prepared in a physiologically acceptable vehicle or carrier. Forsolutions or emulsions, suitable carriers include, for example, aqueousor alcoholic/aqueous solutions, emulsions or suspensions, includingsaline and buffered media. Parenteral vehicles can include sodiumchloride solution, Ringer's dextrose, dextrose and sodium chloride,lactated Ringer's or fixed oils. Intravenous vehicles can includevarious additives, preservatives, or fluid, nutrient or electrolytereplenishers.

A variety of aqueous carriers, e.g., sterile phosphate buffered salinesolutions, bacteriostatic water, water, buffered water, 0.4% saline,0.3% glycine, and the like, and may include other proteins for enhancedstability, such as albumin, lipoprotein, globulin, etc., subjected tomild chemical modifications or the like.

Therapeutic formulations of the antibody are prepared for storage bymixing the antibody having the desired degree of purity with optionalphysiologically acceptable carriers, excipients or stabilizers(Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)),in the form of lyophilized formulations or aqueous solutions. Acceptablecarriers, excipients, or stabilizers are nontoxic to recipients at thedosages and concentrations employed, and include buffers such asphosphate, citrate, and other organic acids; antioxidants includingascorbic acid and methionine; preservatives (such asoctadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionicsurfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

The formulation herein may also contain more than one active compound asnecessary for the particular indication being treated, preferably thosewith complementary activities that do not adversely affect each other.Such molecules are suitably present in combination in amounts that areeffective for the purpose intended.

The active ingredients may also be entrapped in microcapsule prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsule and poly-(methylmethacylate) microcapsule,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes.

Aqueous suspensions may contain the active compound in admixture withexcipients suitable for the manufacture of aqueous suspensions. Suchexcipients are suspending agents, for example sodiumcarboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose,sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia;dispersing or wetting agents may be a naturally-occurring phosphatide,for example lecithin, or condensation products of an alkylene oxide withfatty acids, for example polyoxyethylene stearate, or condensationproducts of ethylene oxide with long chain aliphatic alcohols, forexample heptadecaethyl-eneoxycetanol, or condensation products ofethylene oxide with partial esters derived from fatty acids and ahexitol such as polyoxyethylene sorbitol monooleate, or condensationproducts of ethylene oxide with partial esters derived from fatty acidsand hexitol anhydrides, for example polyethylene sorbitan monooleate.The aqueous suspensions may also contain one or more preservatives, forexample ethyl, or n-propyl, p-hydroxybenzoate.

The antibodies of the present disclosure can be lyophilized for storageand reconstituted in a suitable carrier prior to use. This technique hasbeen shown to be effective with conventional immunoglobulins. Anysuitable lyophilization and reconstitution techniques can be employed.It will be appreciated by those skilled in the art that lyophilizationand reconstitution can lead to varying degrees of antibody activity lossand that use levels may have to be adjusted to compensate.

Dispersible powders and granules suitable for preparation of an aqueoussuspension by the addition of water provide the active compound inadmixture with a dispersing or wetting agent, suspending agent and oneor more preservatives. Suitable dispersing or wetting agents andsuspending agents are exemplified by those already mentioned above.

The concentration of antibody in these formulations can vary widely, forexample from less than about 0.5%, usually at or at least about 1% to asmuch as 15 or 20% by weight and will be selected primarily based onfluid volumes, viscosities, etc., in accordance with the particular modeof administration selected. Thus, a typical pharmaceutical compositionfor parenteral injection could be made up to contain 1 ml sterilebuffered water, and 50 mg of antibody. A typical composition forintravenous infusion could be made up to contain 250 ml of sterileRinger's solution, and 150 mg of antibody. Actual methods for preparingparenterally administrable compositions will be known or apparent tothose skilled in the art and are described in more detail in, forexample, Remington's Pharmaceutical Science, 15th ed., Mack PublishingCompany, Easton, Pa. (1980). An effective dosage of antibody is withinthe range of 0.01 mg to 1000 mg per kg of body weight peradministration.

The pharmaceutical compositions may be in the form of a sterileinjectable aqueous, oleaginous suspension, dispersions or sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersions. The suspension may be formulated according tothe known art using those suitable dispersing or wetting agents andsuspending agents which have been mentioned above. The sterileinjectable preparation may also be a sterile injectable solution orsuspension in a non-toxic parenterally-acceptable diluent or solvent,for example as a solution in 1,3-butane diol. The carrier can be asolvent or dispersion medium containing, for example, water, ethanol,polyol (for example, glycerol, propylene glycol, and liquid polyethyleneglycol, and the like), suitable mixtures thereof, vegetable oils,Ringer's solution and isotonic sodium chloride solution. In addition,sterile, fixed oils are conventionally employed as a solvent orsuspending medium. For this purpose any bland fixed oil may be employedincluding synthetic mono- or diglycerides. In addition, fatty acids suchas oleic acid find use in the preparation of injectables.

In all cases the form must be sterile and must be fluid to the extentthat easy syringability exists. The proper fluidity can be maintained,for example, by the use of a coating, such as lecithin, by themaintenance of the required particle size in the case of dispersion andby the use of surfactants. It must be stable under the conditions ofmanufacture and storage and must be preserved against the contaminatingaction of microorganisms, such as bacteria and fungi. The prevention ofthe action of microorganisms can be brought about by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In manycases, it will be desirable to include isotonic agents, for example,sugars or sodium chloride. Prolonged absorption of the injectablecompositions can be brought about by the use in the compositions ofagents delaying absorption, for example, aluminum monostearate andgelatin.

Compositions useful for administration may be formulated with uptake orabsorption enhancers to increase their efficacy. Such enhancers includefor example, salicylate, glycocholate/linoleate, glycholate, aprotinin,bacitracin, SDS, caprate and the like. See, e.g., Fix (J. Pharm. Sci.,85:1282-1285 (1996)) and Oliyai and Stella (Ann. Rev. Pharmacol.Toxicol., 32:521-544 (1993)).

Antibody compositions contemplated for use to inhibit target activity,including binding of the target to its cognate receptor or ligand,target-mediated signaling, and the like. In particular, the compositionsexhibit inhibitory properties at concentrations that are substantiallyfree of side effects, and are therefore useful for extended treatmentprotocols. For example, co-administration of an antibody compositionwith another, more toxic, cytotoxic agent can achieve beneficialinhibition of a condition or disorder being treated, while effectivelyreducing the toxic side effects in the patient.

In addition, the properties of hydrophilicity and hydrophobicity of thecompositions contemplated for use in the present disclosure are wellbalanced, thereby enhancing their utility for both in vitro andespecially in vivo uses, while other compositions lacking such balanceare of substantially less utility. Specifically, compositionscontemplated for use in the disclosure have an appropriate degree ofsolubility in aqueous media which permits absorption and bioavailabilityin the body, while also having a degree of solubility in lipids whichpermits the compounds to traverse the cell membrane to a putative siteof action. Thus, antibody compositions contemplated are maximallyeffective when they can be delivered to the site of target antigenactivity.

Administration and Dosing

In one aspect, methods of the present disclosure include a step ofadministering a pharmaceutical composition. In certain embodiments, thepharmaceutical composition is a sterile composition.

Methods of the present disclosure are performed using anymedically-accepted means for introducing a therapeutic directly orindirectly into a mammalian subject, including but not limited toinjections, oral ingestion, intranasal, topical, transdermal,parenteral, inhalation spray, vaginal, or rectal administration. Theterm parenteral as used herein includes subcutaneous, intravenous,intramuscular, and intracisternal injections, as well as catheter orinfusion techniques. Administration by, intradermal, intramammary,intraperitoneal, intrathecal, retrobulbar, intrapulmonary injection andor surgical implantation at a particular site is contemplated as well.

In one embodiment, administration is performed at the site of a cancer,fibrosis or affected tissue needing treatment by direct injection intothe site or via a sustained delivery or sustained release mechanism,which can deliver the formulation internally. For example, biodegradablemicrospheres or capsules or other biodegradable polymer configurationscapable of sustained delivery of a composition (e.g., a solublepolypeptide, antibody, or small molecule) can be included in theformulations of the disclosure implanted near or at site of the cancer,fibrosis or affected tissue or organ.

Therapeutic compositions may also be delivered to the patient atmultiple sites. The multiple administrations may be renderedsimultaneously or may be administered over a period of time. In certaincases it is beneficial to provide a continuous flow of the therapeuticcomposition. Additional therapy may be administered on a period basis,for example, hourly, daily, weekly, every 2 weeks, every 3 weeks,monthly, or at a longer interval.

Also contemplated in the present disclosure is the administration ofmultiple agents, such as an antibody composition in conjunction with asecond agent as described herein, including but not limited to achemotherapeutic agent or an agent useful to treat fibrosis.

The amounts of antibody composition in a given dosage may vary accordingto the size of the individual to whom the therapy is being administeredas well as the characteristics of the disorder being treated. Inexemplary treatments, it may be necessary to administer about 1 mg/day,5 mg/day, 10 mg/day, 20 mg/day, 50 mg/day, 75 mg/day, 100 mg/day, 150mg/day, 200 mg/day, 250 mg/day, 500 mg/day or 1000 mg/day. Theseconcentrations may be administered as a single dosage form or asmultiple doses. Standard dose-response studies, first in animal modelsand then in clinical testing, reveal optimal dosages for particulardisease states and patient populations.

It will also be apparent that dosing may be modified if traditionaltherapeutics are administered in combination with therapeutics of thedisclosure.

Kits

As an additional aspect, the disclosure includes kits which comprise oneor more compounds or compositions packaged in a manner which facilitatestheir use to practice methods of the disclosure. In one embodiment, sucha kit includes a compound or composition described herein (e.g., acomposition comprising a target-specific antibody alone or incombination with a second agent), packaged in a container such as asealed bottle or vessel, with a label affixed to the container orincluded in the package that describes use of the compound orcomposition in practicing the method. Preferably, the compound orcomposition is packaged in a unit dosage form. The kit may furtherinclude a device suitable for administering the composition according toa specific route of administration or for practicing a screening assay.Preferably, the kit contains a label that describes use of the antibodycomposition.

Additional aspects and details of the disclosure will be apparent fromthe following examples, which are intended to be illustrative ratherthan limiting.

EXAMPLES Example 1 Isolation of Anti-TGFβ Antibodies from Antibody PhageDisplay Libraries

To isolate a panel of antibodies able to neutralize the activity ofhuman TGFβ, three isoforms of the TGFβ protein, TGFβ1, TGFβ2 and TGFβ3were used for panning of human antibody phage display libraries asdescribed below.

Panning:

The TGFβ antigens (PeproTech, Rocky Hill, N.J. #100-21, 100-35B,100-36E) were first prepared by biotinylating with NHS-PEG4-Biotin(Pierce, Rockford, Ill.) using the manufacturer's protocol. Briefly, theTGFβ antigens, which were stored in low pH buffer, were neutralized byaddition of 20× PBS to bring pH to roughly 6.0. A 30-fold molar excessof the above pre-activated biotin was added and mixed, then kept at roomtemperature for 20 minutes. Then equal volume of 10 mM Glycine pH 3.0was added and the samples were put immediately into dialysis using a 6-8kDa cut-off dialysis unit against a 10 mM Citrate buffer, pH 3.5. A Fabphage display library (XOMA, Berkeley, Calif.) was panned with thebiotinylated TGFβ using a soluble panning method. Each TGFβ isoform waspanned separately in three selection rounds. Kappa and lambdasublibraries were panned separately.

For the first round of phage panning, 50× library equivalents (˜2×10¹²cfu) of the library was blocked on ice for 1 hr in 1 mL of 5% milk/PBS.Binders to streptavidin were deselected from blocked phage by addingblocked phage to streptavidin-coated magnetic DYNABEADS® M-280 andincubating with rotation for 30 minutes. The deselection step wasrepeated once more. A magnet was used to separate beads from phage.Concurrent to the deselection steps, 200 pmoles of biotinylated TGFβ wasallowed to bind streptavidin-coated magnetic DYNABEADS® M-280 byincubating at room temperature with rotation for 30 minutes. Afterbinding, the biotinylated TGFβ beads were washed twice with 5% Milk-PBS.Selection was done by adding deselected phage to biotinylated TGFβ boundto magnetic streptavidin beads and incubating with rotation for 1.5 to 2hours. After selection, unbound phage was washed from beads using aKingfisher magnetic particle processor (Thermo Scientific) which wasprogrammed to wash beads quickly 3 times with PBS-0.1% TWEEN followed byan additional 3 quick washes with PBS. Bound phage was eluted from beadsafter the wash step by the addition of 100 mM triethylamine andincubating with rotation at room temperature for 30 minutes. Elutedphage was neutralized with the addition of equal volume 1M Tris-HCl, pH7.4. Eluted neutralized phage was then collected into a 50 mL Falcontube (Falcon No 352070) and used to infect log growing TG1 bacterialcells (OD₆₀₀˜0.5). Infection was at 37° C. for 30 min without shaking,followed by 30 min additional incubation at 37° C. with shaking at 90rpm. Cells were plated on 2YT media supplemented with 100 ug/mLCarbenicillin and 2% Glucose (2YTCG) agar bioassay plates and incubatedovernight at 30° C. to allow for overnight lawn growth.

In preparation for use as input for the next round, 100× of previousround output was rescued by superinfection using MK07 helper phage. Thiswas done by inoculating 2YTCG media with cells scraped from previouspanning round output. OD_(600 nm) was measured for starting culture andadjusted to reflect a starting OD_(600 nm) of ˜0.05. Cells were grown at37° C. with shaking until cells reached log-growing phase ofOD_(600 nm)˜0.5. Cells were infected with MK07 (New England Biolabs, MA)at a multiplicity of infection (MOI)=˜20, at 37° C. for 30 min withoutshaking, followed by an additional 30 min incubation at 37° C. withshaking at 150 rpm. After infection at 37° C., cells were pelleted andtransferred to new 2YT media supplemented with 50 ug/mL Kanamycin and100 ug/mL Carbenicillin (2YTCK). Cultures were grown overnight at 25° C.Phage was separated from cells and debris by centrifugation andresulting supernatant was recovered and used as input for the nextpanning round. Selection enrichment was monitored by the amount of inputused for each panning round and the resulting phage output titer.

For the second and third panning rounds, the same solution phaseprotocols followed in round one were used with the following exceptions.Phage input amount used in panning rounds two and three was ˜1.0×10¹¹cfu. For round two, 100 pmoles of biotinylated antigen was used inselection, and for round three, 50 pmoles of biotinylated antigen wasused. The Kingfisher was used to wash unbound phage from beads afterselections. In round two, the Kingfisher was programmed to wash beads 3times with PBS-0.1% TWEEN for 2 minutes followed by 1 ml PBS wash for 2minutes repeated 3 times. In round three panning, beads were washed 3times with PBS-0.1% TWEEN for 6 minutes, followed by two four minutewashes and one six minute wash with PBS.

Bacterial periplasmic extracts containing secreted antibody fragmentsfor use in screening for TGFβ binders were prepared by standard methods.Individual colonies were picked into 96 well plates filled with 2YTCsupplemented with 100 ug/mL Carbenicillin and 0.1% glucose media.Cultures were allowed to grow at 37° C. with shaking until log growingphase was reached (OD_(600 nm)=0.5). Colonies were then induced toproduce soluble fragment antibodies by adding 1 mM IPTG final andincubated overnight at 25° C. with shaking. Periplasmic extracts (PPE)containing soluble fragment antibodies were prepared from the inducedcells using the standard method of adding 1:3 volume ratio of ice-coldPPB solution (Teknova, Hollister, Calif.) and double distilled water(ddH₂O) with complete EDTA free protease inhibitor cocktail tablets. PPEwere then used to screen for TGF-β binders.

Screening:

Two alternative screening assay formats were used to identify clonesthat bound TGFβ, including clones that bound to all three TGFβ isoformsand were unique in their sequences. The first screening assay used aplate-based immune-assay and the other screening assay was performedusing an SPR screening method. The plate-based assay involved coatingopaque 384 well white EIA plates with 1 ug/mL Anti-His antibody cloneAD.1.10 (R&D Systems, Minneapolis, Minn.) at 1 ug/mL in PBS buffer forfour hours at room temperature. Then the plate was washed 3× inPBS-TWEEN and then blocked with 0.5% BSA in PBS-TWEEN for 1 hour at roomtemperature. Next 30 uL/well of biotinylated TGFβ was added at between0.1 ug/mL for TGFβ1 and TGFβ2, and 0.2 ug/mL for TGFβ3, diluted inblocking buffer. Then 30 uL of periplasmic extract was added andincubated at 4° C. overnight on gentle plate shaker. Plates were washed3× in PBS-TWEEN then added 50 uL/well of 2.5 ug/mL Streptavidin-Europium(SA-Eu, PerkinElmer) diluted in DELFIA assay dilution buffer(PerkinElmer) to each well and incubated at room temp for 30 minutes ona shaker. Plates were washed 7 times with PBS-TWEEN and added 50uL/wellof the DELFIA enhancement reagent (PerkinElmer) and put on shaker for 8minutes at room temperature then read on Molecular Devices FlexStation 3plate reader in TRF mode with 200-1200 μs collection time and Exc.=345nm, Emm.=618 nm, and cutoff=590 nm, High PMT setting, 20 Reads/well.Samples with signal of more than 2.1-fold higher signal than negativePPE control were considered to be positive.

The SPR assay was performed by a BIACORE A100 direct binding assay. Inthis assay, a CM5 BIACORE chip was prepared via standard amine couplingchemistry using the BIACORE Amine Coupling kit (GE Healthcare,Piscataway, N.J.). The TGFβ antigens were diluted to 6 ug/mL in acetatepH 4.0 and injected for 7 minutes (spots 1, which is TGFβ1) and 10minutes (spots 2 and 4, which are TGFβ2, and TGFβ3). This immobilizesbetween 3400 and 4800 RU of each TGFβ antigen. Samples were deactivatedwith 1M ethanolamine. Periplasmic extracts were diluted 1:1 with HBS-EP+(Teknova) with 2 mg/mL BSA and filtered through a 0.2 uM Millex GVfilterplate (Millipore) and then injected at 30 uL/minute for 240seconds with a 30 second dissociation. Regeneration after each PPEinjection was 10 seconds of 100 mM HCl. The stability early report pointin the BIACORE A100 software was used to evaluate PPE binding levels.Cut-off levels were determined for each TGFβ isoform independently asbeing visually above background level. RU cutoffs were 245, 175, and 125for TGFβ1, TGFβ2 and TGFβ3, respectively.

Affinity Maturation:

One antibody, XPA.42.068, which had significantly greater binding andneutralizing activity for TGFβ1 and TGFβ2 relative to TGFβ3, wassubjected to affinity maturation to increase its affinity and potencyagainst TGFβ3. A library of sequence variants generated from affinitymaturation was panned using TGFβ2 and TGFβ3, with output clones screenedprimarily for improved TGFβ3 binding.

For screening, the SPR assay was performed by a BIACORE A100 directbinding assay. In this assay a CM5 BIACORE chip was prepared viastandard amine coupling chemistry using the BIACORE Amine Coupling kit.The TGFβ antigens were diluted to 1 ug/mL in acetate pH4.0 and injectedfor 5 minutes (spots 1 and 5, which are TGFβ3 and TGFβ1 respectively)and 8 minutes (spots 2, which is TGFβ2). This immobilizes between 200and 450 RU of each TGFβ. Samples were deactivated with 1M ethanolamine.Periplasmic extracts were diluted 1:1 with HBS-EP+ with 2 mg/mL BSA andfiltered through a 0.2 μm Millex GV filter plate (Millipore) and theninjected at 30 uL/minute for 240 seconds with a 600 second dissociation.Regeneration after each PPE injection was 10 seconds of 100 mM HCl.Reference subtracted data was plotted and examined visually for clonesthat appeared to have either greater stability or higher binding levels.One derivative clone, designated XPA.42.681, which demonstrated enhancedbinding to TGFβ3, was included in further characterization studies.

Selected clones were reformatted as IgG2 antibodies. The variable heavy(VH) and light (VL) chains of the selected Fab fragments werePCR-amplified, cloned into plasmid vectors containing antibody constantregion sequences, and transiently transfected into 293E cells usingstandard methods to generate material for further characterization,including the studies described below.

Example 2 Measurement of Binding Affinities of TGFβ Antibodies

Antibodies were characterized against TGFβ isoforms TGFβ1, TGFβ2, andTGFβ3 for their binding affinity (KD), off-rate (kd) and on-rate (ka)using surface plasmon resonance (SPR) technology. The analysis wasperformed using two methods. One method was an antigen directimmobilization method in which the TGFβ proteins were immobilized to asurface at low density with the antibodies injected at multipleconcentrations for kinetic analysis. The other method was an immobilizedantibody method using injections of various concentrations of injectedTGFβ proteins.

Immobilized Antibody Kinetics Method:

A CM4 sensor chip (GE Healthcare) was used on a BIACORE 2000 system (GEHealthcare). The chip was preconditioned with two 30 second injectionseach at 50 μL/minute flow rate of 100 mM HCl, Glycine pH 2.0, 50 mMNaOH, and running buffer prior to immobilization. Running buffer forimmobilization was a HEPES Buffered Saline (HBS-EP+) with 10 mM Hepes,150 mM Sodium Chloride, 3 mM EDTA, and 0.05% Polysorbate 20 (Teknova).The chip surface was activated with a seven minute injection at 10μL/minute of a freshly mixed 1:1 solution of 0.1 M N-Hydroxysuccinimide(NHS) and 0.4 M 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimidehydrochloride (EDC). Following the activation injection, 1 ug/mLanti-TGFβ antibody in acetate pH 4.5 was injected at 10 μL/minute forone minute, with injections targeting 120 RU. 8 minutes of 1MEthanolamine hydrochloride-NaOH pH 8.5 was injected to block thesurface. The NHS, EDC, and Ethanolamine used were from the BIACORE AmineCoupling Kit.

Kinetic Analysis was performed using a running buffer of thoroughlydegassed form of the HBS-EP+ buffer above supplemented with 1 mg/mL BSA(Sigma Aldrich, St. Louis, Mo.). TGFβ sample injections were performedat 50 μL/minute for four minutes with a 900 second dissociation time.Each TGFβ protein (TGFβ1, TGFβ2, TGFβ3) was injected at 10 nM, 2 nM, 0.4nM, 0.08 nM (350 ng/mL with 5 fold serial dilution) with blanksbracketing each concentration series and quadruplicate injections.Regeneration was then performed with three injections of 30 seconds eachof 100 mM HCl in 3 M MgCl₂ followed by a final 30 second blank bufferinjection.

The data were analyzed using Scrubber2 (BioLogic Software, CampbellAustralia) and was double referenced by subtracting both the blank flowcell data and the averaged bracketing blank injections. The data was fitby simultaneously fitting the (KD) an off-rate (kd) and on-rate (ka),and are shown in Table 2 below. Data for a previously measuredcomparator antibody, designated BM-1 (1D11, R&D Systems MAB1835) alsoare included in Table 2. BM-1 data was generated on the BIACORE A100.Briefly the BM-1 antibody was captured at approximately 100 RU densityon a high density Rabbit anti-mouse Fc CM5 chip surface (GE Healthcare).TGFβ proteins were injected at the same concentrations as describedabove at 30 μL/minute. These data were double referenced and analyzed inBIACORE A100 software.

TABLE 2 Affinity data from assay utilizing immobilized antibody andinjected TGFβ TGFβ1 TGFβ2 TGFβ3 Antibody ka (1/Ms) kd (1/s) KD ka (1/Ms)kd (1/s) KD ka (1/Ms) kd (1/s) KD XPA.42.068 1.53E+07 9.05E−04 59 pM1.04E+07 5.35E−04 51 pM 8.45E+06 3.84E−03 455 pM XPA.42.089 4.40E+071.67E−04 3.8 pM 1.62E+07 4.14E−04 25 pM 7.70E+06 1.09E−02 1.4 nMXPA.42.681 4.25E+07 7.20E−05 1.7 pM 1.71E+07 4.99E−05 2.9 pM 1.30E+077.50E−05 5.7 pM BM-1 1.90E+07 1.40E−03 72 pM 1.10E+07 2.00E−03 170 pM6.50E+06 3.10E−04 48 pM

The affinity data as measured in this assay using immobilized antibodiesshowed that XPA.42.681 had the strongest (tightest) binding of any ofthe antibodies for each of the three isoforms of TGFβ, and also boundeach of the TGFβ isoforms with similar affinities. In addition, theantibodies XPA.42.068 and XPA.42.089 had similar or stronger binding tothe TGFβ1 and TGFβ2 isoforms compared with the BM-1 antibody, but showedsignificantly less binding to the TGFβ3 isoform, compared either to theBM-1 antibody or relative to TGFβ1 and TGFβ2 binding.

Immobilized TGFβ Affinity Method:

A CM1 sensor chip (GE Healthcare) which has a planar —COOH surface wasused on a BIACORE 2000 system. The chip was preconditioned with two 30second injections each at 50 μL/minute flow rate of 100 mM HCl, GlycinepH 2.0, 50 mM NaOH, 1% SDS, and running buffer prior to immobilization.Running buffer for immobilization was a HEPES Buffered Saline (HBS-EP+)with 10 mM Hepes, 150 mM Sodium Chloride, 3 mM EDTA, and 0.05%Polysorbate 20. The chip surface was activated with four minuteinjections at 20 μL/minute of a freshly mixed 1:1 solution of 0.1MN-Hydroxysuccinimide (NHS) and 0.4M 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC). Following the activation injection a0.1 ug/mL solution of TGFβ in acetate pH 4.0 was injected at 20μL/minute for several minutes. Each TGFβ utilized a separate activationstep on its own flow cell such that TGFβ1 was immobilized on Fc2, TGFβ2on Fc3, and TGFβ3 on Fc4 with Fc1 as an activated and inactivated blank.Injections of TGFβ were performed as a series of 1 to 2 minuteinjections looking at immobilized level between each injection. Thetarget immobilized density of each TGFβ ligand was 30 RU. After the TGFβimmobilization injections, 4 minutes of 1 M Ethanolaminehydrochloride-NaOH pH 8.5 was injected to block the surface. The NHS,EDC, and Ethanolamine used were from the BIACORE Amine Coupling Kit andthe TGFβ1, TGFβ2, and TGFβ3 were from R&D Systems.

For affinity analysis the running buffer was switched to a thoroughlydegassed form of the HBS-EP+ buffer above supplemented with 1 mg/mL BSA(Sigma Aldrich, St. Louis, Mo.). Each of the antibodies was diluted inrunning buffer to 5 μg/mL (33.3 nM) and 4 subsequent five-fold dilutionswere prepared setting up concentrations of 33.33 nM, 6.67 nM, 1.33 nM,267 pM, and 53 pM for each. These were then injected using the Kinjectsetting for four minutes at 50 μL/minute, with a 900 second dissociationtime. Regeneration was then performed with a 12 μL (14.4 second)injection of 100 mM HCl at 50 μL/minute followed by an 18 second bufferinjection. Injections were across all flow cells simultaneously andsamples were run injected in quadruplicates with blank injectionsbracketing each set of descending concentration injection groups foreach antibody. This means that before the same sample was injected asecond time all other concentrations of all antibodies were injectedonce.

The data were analyzed using Scrubber2 (BioLogic Software, CampbellAustralia) and were double referenced by subtracting both the blank flowcell data and the averaged bracketing blank injections. The data werefit by simultaneously fitting the (KD) an offrate (kd) and onrate (ka),and are shown in Table 3 below.

TABLE 3 Affinity data from assay utilizing immobilized TGFβ and injectedantibodies. TGFβ1 TGFβ2 TGFβ3 Antibody ka (1/Ms) kd (1/s) KD ka (1/Ms)kd (1/s) KD ka (1/Ms) kd (1/s) KD XPA.42.068 5.44E+06 1.70E−03 313 pM7.30E+06 7.98E−04 109 pM 5.45E+06 6.96E−03 1.3 nM XPA.42.089 5.98E+061.06E−03 177 pM 4.80E+06 1.39E−03 290 pM 3.40E+06 5.70E−02 >17 nMXPA.42.681 1.14E+07 3.63E−04  32 pM 1.28E+07 3.94E−04  31 pM 1.23E+076.65E−04 54 pM BM-1 1.28E+07 3.90E−03 304 pM 7.00E+06 6.84E−03 977 pM5.05E+06 9.46E−04 188 pM

Consistent with the immobilized antibody results from Table 2, theaffinity data measured in assays using immobilized antigen (Table 3)also showed that XPA.42.681 had the strongest (tightest) binding of anyof the antibodies for each of the three isoforms of TGFβ, with similaraffinity for each of the TGFβ isoforms. In addition, XPA.42.068 andXPA.42.089 had similar or stronger binding to the TGFβ1 and TGFβ2isoforms compared with BM-1, but significantly less binding to the TGFβ3isoform, compared either to the BM-1 antibody or relative to TGFβ1binding. The difference in rate constants measured using the immobilizedantibody versus immobilized antigen assays likely results from theinherent complexities of the system, but nevertheless each providesrelatively high quality kinetic data and consistency in bindingproperties across the TGFβ isoforms and among the antibodies relative toeach other.

Example 3 Measurement of Receptor Competition by TGFβ Antibodies

Antibodies were characterized for their ability to inhibit or block thebinding of each of the three TGFβ ligands to TGFβ receptors by SPRcompetition assays. TGFβ signals through the TGFβ type II receptor(TGFβ-RII) which is a serine threonine kinase transmembrane protein andrequires the cytoplasmic association of the TGFβ receptor type 1 protein(TGFβ-R1) for activation. The ligand binding role of TGFβ-RI is notclear and a recombinant form of TGFβ-RI did not demonstrate any bindingat tested concentrations to any of the TGFβ1, TGFβ2 or TGFβ3 ligands, orthe TGFβ-RII bound forms of those ligands, and therefore could not beevaluated in receptor competition experiments. The TGFβ type IIIreceptor (TGFβ-RIII) has both membrane bound and soluble forms and isnot believed to be involved in TGFβ signaling. The TGFβ-RIIb is a splicevariant that contains a 26 amino acid insertion near the N-terminus andhas the unique property of binding to all three of the TGFβ isoformswith good affinity. The TGFβ-RII binds tightly only to TGFβ1 and TGFβ3ligands, while the TGFβ-RIII binds best to TGFβ2 ligand.

A CM5 sensor chip (GE Healthcare) was used on a BIACORE 2000 system. Thechip was preconditioned with several 30 second injections each at 50μL/minute flow rate of 100 mM HCl and 50 mM NaOH prior toimmobilization. Running buffer for immobilization was a HEPES BufferedSaline (HBS-EP+) with 10 mM Hepes, 150 mM Sodium Chloride, 3 mM EDTA,and 0.05% Polysorbate 20. The chip surface was activated with a sevenminute injection at 10 μL/minute of a freshly mixed 1:1 solution of 0.1M N-Hydroxysuccinimide (NHS) and 0.4 M 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC). Following the activation injection, 5ug/mL TGFβ-RII, TGFβ-RIIb, or TGFβ-RIII (R&D Systems) in acetate pH 4.5was injected at 20 μL/minute for four minutes and resulted in 1000-4000RU immobilized for each of the TGFβ receptors. Then, 8 minutes of 1 MEthanolamine hydrochloride-NaOH pH 8.5 was injected to block thesurface. The NHS, EDC, and Ethanolamine used were from the BIACORE AmineCoupling Kit. Fc1 was the activated and deactivated control.

Competition assays were performed using a running buffer of thoroughlydegassed form of the HBS-EP+ buffer above supplemented with 1 mg/mL BSA.TGFβ ligands were used in all injections except blank controls at 100ng/mL (10 nM) to 40 ng/mL (4 nM) and were prepared with 10 ug/mL (66.6nM) of competitor and control antibodies. Samples were allowed to cometo equilibrium for 40 minutes at room temperature before the BIACORE runwas started. Equilibrated samples were then injected at 10 uL/minute fortwo minutes. Regeneration was performed every cycle with one injectionof pH 2.5 glycine at 50 uL/minute for 9.6 seconds (8 μLs). Samples wererun in at least duplicates and analyzed for the level of TGFβ bound.

As shown in Table 4 below, the results for antibodies XPA.42.068,XPA.42.089, XPA.42.681 and the BM-1 comparator suggest that each ofthese antibodies blocks the association of all three TGFβ ligands to theTGFβ-RII and TGFβ-RIII receptors, and that no clear distinction wasmade. This receptor competition pattern was not universal among all ofthe other antibodies tested, but for which data is not shown in thepresent disclosure.

TABLE 4 Receptor competition assay EC50 (nM antibody) XPA.42.068XPA.42.089 XPA.42.681 BM-1 TGFβ1/TGFβ-RII 2.0E−09 1.7E−09 2.3E−092.4E−09 TGFβ2/TGFβ-RII 2.2E−09 1.8E−09 1.7E−09 2.8E−09 TGFβ3/TGFβ-RII1.4E−09 3.4E−08 1.2E−09 1.6E−09 TGFβ1/TGFβ- 6.0E−10 1.2E−09 2.2E−092.1E−09 RIII TGFβ2/TGFβ- 2.4E−09 1.6E−09 2.1E−09 2.5E−09 RIIITGFβ3/TGFβ- 1.9E−09 3.1E−08 1.1E−09 1.4E−09 RIII

The potency of the XPA.42.068, XPA.42.089, XPA.42.681 and BM-1antibodies in receptor competition generally correlated with theiraffinities to the various isoforms of TGFβ.

Example 4 Measurement of Epitope Competition Among TGFβ Antibodies

The ability of the XPA.42.068 and XPA.42.089 antibodies to bind toindependent or overlapping epitopes on the TGFβ proteins was evaluated.While this pair-wise analysis was not straightforward due to varyingaffinities of the antibodies among the different isoforms of TGFβ, andthe covalent homodimerization of TGFβ ligands, which results in bindingin a two IgG per homodimer ratio (e.g., self pairing), a solublecompetition-based assay was developed.

A CM5 sensor chip (GE Healthcare) was used on a BIACORE 2000 system. Thechip was preconditioned with four 30 second injections at 50 uL/minuteflow rate of 100 mM HCl prior to immobilization. Running buffer forimmobilization was a HEPES Buffered Saline (HBS-EP+) with 10 mM Hepes,150 mM Sodium Chloride, 3 mM EDTA, and 0.05% Polysorbate 20. The chipsurface was activated with a seven minute injection at 10 uL/minute of afreshly mixed 1:1 solution of 0.1 M N-Hydroxysuccinimide (NHS) and 0.4 M1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC).Following the activation injection, a 1 ug/mL antibody XPA.42.089 inacetate pH 4.5 and was injected at 10 μL/minute for several minutesinjections. Injections were monitored and performed sequentially toestablish immobilization levels very close to 300 RU. Eight minutes of 1M Ethanolamine hydrochloride-NaOH pH 8.5 was injected to block thesurface. The NHS, EDC, and Ethanolamine used were from the BIACORE AmineCoupling Kit. Fc1 was the activated and deactivated control.

Competition assays were performed using a running buffer of thoroughlydegassed form of the HBS-EP+ buffer above supplemented with 1 mg/mL BSA.TGFβ1, TGFβ2 and TGFβ3 were used in all injections at 0.1 ug/mL (4 nM),except blank controls, and were prepared with 20 ug/mL (133 nM) ofcompetitor antibodies. The TGFβ-RIIb-Fc recombinant receptor (R&DSystems) also was included as a competitor. Samples were allowed to cometo equilibrium for 40 minutes at room temperature before the BIACORE runwas started. Equilibrated samples were then injected at 30 uL/minute forthree minutes over all of the flow cells. Regeneration was performedevery cycle with one injection of 50 mM NaOH at 50 uL/minute for 6seconds (5 μLs) and followed by a thirty second buffer injection.Samples were run in duplicates and analyzed for level of TGFβ bound atthe end of the three minutes.

TABLE 5 Binding competition (XPA.42.089 immobilized) TGFβ1 TGFβ2 TGFβ3Blank −0.568 0.0655 0.3684 No Ab 63.85 61.05 23.65 XPA.42.068 10.85 5.187.32 XPA.42.089 1.7 0.3635 9.77 TGFβ-RIIb −0.316 38.2 −0.378

As shown in Table 5 above, the data indicate that the XPA.42.068 andXPA.42.089 exhibited strong competition with each other for binding eachof the TGFβ isoforms. The values represent the average RU or signalintensity of TGFβ binding that was measured during the injections ofcomplex. This shows that the signal is greatly reduced when thecomplexed antibody is present. Any dissociation of the complex duringthe injection could allow for free or monovalently bound TGFβ to bebound by the XPA.42.089 capture antibody. It has been shown that theTGFβ-RIIb interaction with TGFβ2 is much weaker than the TGFβ1 and TGFβ3proteins and the rapid offrate allows for relatively poor competitionfor TGFβ2 against the high affinity XPA.42.089. The XPA.42.681 antibody,which was derived from XPA.42.068, was not tested in the competitionassays.

Example 5 Measurement of rhLAP Competition by TGFβ Antibodies

Additional competition assays were undertaken to determine whether theantibodies also interact with the latent form of TGFβ. The TGFβpro-protein is cleaved within the golgi by a furin-like convertase intoa N-terminal 249 amino acid latency associated peptide and a C-terminal112 amino acid mature TGFβ1.

A CM5 sensor chip (GE Healthcare) was used on a BIACORE 2000 system. Thechip was preconditioned with several 30 second injections each at 50μL/minute flow rate of 100 mM HCl and 50 mM NaOH prior toimmobilization. Running buffer for immobilization was a HEPES BufferedSaline (HBS-EP+) with 10 mM Hepes, 150 mM Sodium Chloride, 3 mM EDTA,and 0.05% Polysorbate 20. The chip surface was activated with a sevenminute injection at 10 μL/minute of a freshly mixed 1:1 solution of 0.1M N-Hydroxysuccinimide (NHS) and 0.4 M 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC). Following the activation injection 2ug/mL recombinant human TGFβ1 Latency Associated Peptide (rhLAP) (R&DSystems) in acetate pH 4.5 was injected at 10 μL/minute for four minutesand resulted in 400 RU of rhLAP immobilized. 8 minutes of 1 MEthanolamine hydrochloride-NaOH pH 8.5 was injected to block thesurface. Fc1 was the activated and deactivated control.

The rhLAP competition assay was performed using a running buffer of athoroughly degassed HBS-EP+ buffer as above supplemented with 1 mg/mLBSA. TGFβ1 was used in all injections except blank controls at 0.25ug/mL (10 nM) and was prepared with 10 ug/mL (66.6 nM) of competitor andcontrol antibodies. Samples were allowed to come to equilibrium for 40minutes at room temperature before starting the BIACORE run.Equilibrated samples were then injected at 40 uL/minute for two minutesover the control and the rhLAP surface. Regeneration was performed everycycle with two injections of 100 mM HCl at 100 uL/minute for 9.6 seconds(16 μLs). Samples were run in duplicates and analyzed for the level ofTGFβ1 bound.

The antibodies XPA.42.068, XPA.42.089 and BM-1 comparator were eachtested in the rhLAP competition assay. The XPA.42.681 antibody, whichwas derived from XPA.42.068, was not tested. As shown in FIG. 1,XPA.42.068, XPA.42.089 and BM-1 each exhibited a high level ofcompetition with rhLAP, indicating that the antibodies interact with theactive form of TGFβ and do not recognize latent TGFβ.

Example 6 Measurement of Neutralization by TGFβ Antibodies in HT-2 Assay

To determine if the antibodies functionally neutralized TGFβ isoforms,the assay methods of Ruegemer et al. (J Immunol. 144:1767-76; 1990) wereadapted whereby HT-2 murine T cells are grown with IL-4, and with orwithout the addition of TGFβ1, TGFβ2 or TGFβ3. TGFβ isoforms inhibitIL-4 dependent growth of HT-2 cells through transactivation of genespromoting cell cycle arrest. IL-4 transactivates a mitogenic geneexpression program by activating targets such as c-myc and GM-CSF;whereas TGFβ signaling transactivates genes which suppress c-myc andGM-CSF expression. If TGFβ signaling is abrogated by a neutralizingantibody, HT-2 cells proliferate. Differences in growth were scored byCELL TITERGLO® (Promega #G7571) viability assay which measures ATP as areadout for metabolically active cells.

HT-2 murine T cells were maintained by splitting every 2-3 days at1.5e4-2.5e4 cells/mL in RPMI+10% FBS, 10 mM Hepes, 2 mM glutamine, 50 uM2-ME. Fresh recombinant mouse IL-2 (R&D Systems) was added at 200 IU/mLto each flask from a concentrated stock. On day 1, cells were washed inmedia to remove IL-2 and dispensed into opaque 96 well plates at 10,000cells per well with 2000 IU/ml recombinant mouse IL-4 (R&D Systems).TGFβ1, TGFβ2 or TGFβ3 (PeproTech #100-21, 100-35B, 100-36E) was addedafter 1 hour pre-incubation with or without antibodies across atitration series. After 48 hour incubation at 37° C., viable cellpopulation was scored on MDS Flexstation3 using CELL TITERGLO® accordingto manufacturers recommendations.

TABLE 6 HT-2 cell neutralization assay Antibody TGFβ2 TGFβ1 TGFβ3XPA.42.068 + 98.0 0.0 XPA.42.089 + 57.0 0.0 XPA.42.681 + Potent 30.3BM-1 + 220.0 196.0

The antibodies were initially tested for neutralization of TGFβ2activity at a single 10 ug/ml dilution point in the HT-2 assay, and eachof the antibodies was confirmed to be positive, with the antibodiesXPA.42.068, XPA.42.089 and XPA.42.681 having greater potency than theBM-1 comparator antibody at the single point tested. Neutralization ofTGFβ1 and TGFβ3 was then determined and an IC50 calculated for eachantibody across a 6 point dilution series. Again, each of theXPA.42.068, XPA.42.089 and XPA.42.681 demonstrated greater potency thanthe BM-1 comparator with respect to TGFβ1 neutralization, but onlyXPA.42.681 was found to exhibit greater potency TGFβ3 neutralization,and thus was the most potent pan-inhibitor of TGFβ (Table 6). XPA.42.681exhibited enhanced potency in this assay, with the lowest concentrationstested significantly inhibiting TGFβ1, and thus a specific IC50calculation could not be made.

Example 7 Measurement of Neutralization by TGFβ Antibodies in IL-11Release Assay

A second neutralization assay scored TGFβ mediated secretion of IL-11from A549 lung carcinoma cells, which is part of a pro-fibrotic responsein lung fibroblasts and epithelial cells. TGFβ also mediates secretionof IL-11 from MDA-MB-231 cells which promotes metastasis to the bone.This assay models TGFβ mediated biological responses that contribute tofibrosis and metastatic disease. The IL-11 release assay was adaptedfrom Rapoza et al. (J Immunol. Methods 316:18-26; 2006), whereby A549cells were seeded into 96 well plates and the next day cells weretreated with or without the TGFβ isoforms, pre-incubated with or withoutneutralizing antibodies. IL-11 release was scored in cell culturesupernatants by ELISA.

In this assay, A549 cells were grown in F12+10% serum. The day prior toanalysis, cells were detached with versene (to retain receptorexpression) and seeded at 40,000 cells/well into a 96 well flat bottomplate. The next day TGFβ1, TGFβ2 or TGFβ3 at EC80 was pre-incubated for1 hour with or without antibodies across a dilution series prior toadding to cells. As a control, TGFβ alone, TGFβ+anti-KLH-G2 controlantibody or media alone was added to plates. After 24 hours at 37° C.,supernatant was harvested and IL-11 was scored by ELISA using the IL-11Duo Set ELISA kit (R&D Systems) according to manufacturer'srecommendations.

TABLE 7 IL-11 Release Assay - IC50 (ng/mL) Antibody TGFβ1 TGFβ2 TGFβ3XPA.42.068 220.5 110.5 1795.0 XPA.42.089 37.0 58.0 0.0 XPA.42.681 0.41.0 0.8 BM-1 292.0 498.0 12.0

As shown in Table 7 above, and similar to the HT-2 assay, the IL-11release assay results indicated that XPA.42.681 was the most potent ofany of the antibodies for each of the three isoforms of TGFβ. Incontrast to the HT-2 assay, the XPA.42.681 antibody exhibited a dosedependent effect on IL-11 release which enabled IC50 determination, andalso revealed generally similar IC50 values for each TGFβ isoform.Antibodies XPA.42.068 and XPA.42.089 also showed good neutralization ofthe TGFβ1 and TGFβ2 isoforms (more potent than BM-1 comparator), butwith significantly less neutralization of TGFβ3 compared either to theBM-1 antibody or relative to the neutralization of TGFβ1 and TGFβ2.

Example 8 Measurement of Neutralization by TGFβ Antibodies in pSMAD2Assay

To further characterize the antibodies, a phospho-SMAD2 (pSMAD2) assaywas developed to score neutralization of TGFβ signaling through theTGFβRII/TGFβRI receptor complex. Detroit 562 cells were maintained inIMDM+10% FBS. Cells were detached with versene and plated into a 6 welldish at 500,000 cells per well. The next day, the cells were serumstarved in serum free IMDM for 3 hours prior to 30 minute exposure toTGFβ1, TGFβ2 or TGFβ3 pre-incubated for 1 hour with or withoutantibodies. After 30 minutes at 37° C., cells were lysed and pSMAD2 andtotal SMAD2 was scored by ELISA using commercial kits (Cell SignalingTechnology, Danvers, Mass.) according to the manufacturer'srecommendations for detection. Percentage of pSMAD2 was normalized tototal SMAD2 and percent inhibition was calculated for each clone fromnormalized % pSMAD2 relative to anti-KLH control (FIG. 2). T test (twotailed) showed that the XPA.42.681 antibody was significantly morepotent than the BM-1 comparator antibody in neutralizing pSMAD signalingacross all TGFβ isoforms (p<0.05). Additionally, XPA.42.068 wassignificantly more potent against TGFβ2 relative to the BM-1 comparator.

Example 9 Measurement of TGFβ Antibody Activity in a Regulatory T CellAssay

To characterize the activity of the antibodies on endogenous TGFβ, aregulatory T (Treg) cell assay was established, based on methods similarto Tran et al. (Blood 110:2983-2990; 2007). T cells were isolated fromfrozen vials of human PBMCs using the EasySep T cell Enrichment kit(StemCell Technologies, Vancouver, BC). T cells were activated withplate-bound anti-human CD3 antibody (eBioscience, San Diego, Calif.) at10 ug/ml and soluble anti-human CD28 antibody (eBioscience) at 2 ug/ml.The cells were also treated concurrently with 15 ug/ml of the TGFβantibodies or controls. After 4 days, the cells were stained withanti-human CD4-FITC (BD Biosciences) and anti-human CD25-A647(BioLegend, San Diego, Calif.) for 30 minutes at 4° C. Cells were fixedwith FOXP3 Fix buffer (BioLegend) for 20 minutes at room temperature,and permeabilized for 15 minutes at room temperature with FOXP3permeabilization buffer (BioLegend). Cells were stained with 1:25dilution of anti-human FOXP3-PE (BioLegend) and analyzed on a BDFACSCanto™ system. CD4+ cells were gated and CD4+CD25+Foxp3+ subpopulations were quantitated with Flowjo software. Antibodies wereevaluated in this assay using 4 or 5 different PBMC donors andrepresentative data from 2 donors are shown (FIG. 3).

Although a range of activity was found due to donor dependentdifferences in cell populations, generally, the XPA.42.681 and the BM-1comparator antibodies inhibited the Treg cell population, while theXPA.42.068 and XPA.42.089 antibodies provided partial activity in thisassay.

Example 10 Measurement of TGFβ Antibody Activity in an EMT Assay

Epithelial to mesenchymal transition (EMT) enables self renewal of tumorcells to promote cancer invasion and metastasis. Induction of EMT isdriven by cytokines, including TGFβ1, TGFβ2 and TGFβ3, and all threeisoforms may be involved sequentially in EMT depending on tissue type(Boyer et al., Dev. Biol. 208:530-545, 1999; Bhowmick et al., Mol. Biol.Cell 12:27-36, 2001; Camenisch et al., Dev. Biol. 248:170-181, 2002). AnEMT assay was developed using primary human mammary epithelial cells(HMEC), similar to Mani et al. (Cell 133:704-715; 2008) to determine ifthe antibodies inhibit this process in vitro.

Human mammary epithelial cells (Lonza, Basel, Switzerland) were grown inMEGM complete media (Lonza) as recommended by manufacturer. Forsub-culturing, cells were trypsinized and treated with trypsinneutralizing solution (Lonza) prior to seeding. HMEC cells were seededat 3500 cells/cm² in 8-well chamber slides and treated with or withoutTGFβ at 2.5 ng/ml, pre-incubated with or without antibodies for 30minutes. Cells were incubated at 37° C. for 8 days and freshmedia+reagents were added after 4 days. On day 8, cells were fixed with4% paraformaldehyde for 15 minutes at room temperature. Cells wererinsed twice in PBS and permeabilized in PBS+0.25% Triton X-100 for 10minutes, before blocking with PBS-TWEEN+10% goat serum for 30 minutes.Cells were stained overnight at 4° C. for a mesenchymal marker usinganti-human vimentin (Cell Signaling Technology, Danvers, Mass.) and foran epithelial marker using anti-human E-Cadherin (Cell SignalingTechnology) diluted 1:200 or 1:500, respectively. Cells were washed inPBS 3 times and incubated with appropriate secondary antibodies AlexaFluor 488 goat anti-rabbit or Alexa Fluor 568 goat anti-rabbit(Invitrogen, Carlsbad, Calif.) diluted in blocking solution for 1 hourat room temperature and protected from light. Slides were washed andmounted with Gold Anti-Fade/DAPI prior to fluorescence microscopy.

Exposure of HMEC cells to TGFβ in the presence of the anti-KLH controlantibody results in increased vimentin staining and a reduction in totalcell density, consistent with TGFβ mediated growth arrest anddifferentiation to a mesenchymal phenotype. Neutralization of TGFβ1mediated EMT was evident based on reduced vimentin staining, whichcorrelated with increased cell density for the XPA.42.681, XPA.42.068and XPA.42.089 antibodies, while an intermediate response was observedfor the BM-1 comparator, as vimentin staining was present, although notto the same degree as anti-KLH control (data not shown). Additionally,each of the antibodies inhibited EMT driven by TGFβ2, although the BM-1comparator antibody appeared less potent based on Vimentin signalintensity, E-cadherin staining and increased cell density. Forneutralization of TGFβ3 mediated EMT, the XPA.42.681 antibody was mostpotent, followed by BM-1 and XPA.42.068, while XPA.42.089 did not appeardifferent from the anti-KLH control.

Example 11 Tumor Inhibition by TGFβ Antibodies in a Xenograft MouseModel

The antibodies XPA.42.068 and XPA.42.089 were evaluated for theirability to inhibit tumor growth in a xenograft model derived fromDetroit 562, a human pharyngeal cancer cell line (Van Aarsen et al.,Cancer Res. 68:561-70; 2008). Eight to nine week old Nu/Nu mice (CharlesRiver Laboratories) were implanted subcutaneously with 5×10⁶ Detroit 562cells in BD MATRIGEL™ (1:1, 200 uL) per animal, into the lower leftventral abdominal region. Animals were randomized into test groups oftwelve mice each: anti-KLH human IgG2 isotype control (10 mg/kg),XPA.42.068 (1, 3, or 10 mg/kg dose), XPA.42.089 (1, 3, or 10 mg/kgdose), BM-1 comparator (3 mg/kg), or mouse isotype control IgG1 (3mg/kg). Dosing and tumor volume measurements were done biweekly (FIG.4). Animals were sacrificed the day after the last dose (day 28), after7 doses of antibody treatment. For all measurements, statisticalsignificance was determined by one-tailed Student's t-test.

As shown in FIG. 4, tumors treated with XPA.42.089 trended smaller thantumors treated with XPA.42.068, with significant differences in thehigher dose levels when compared to anti-KLH human IgG2 control. PercentTumor Growth Inhibition (TGI) was compared to IgG control antibody onday 28 in all test groups. Tumors from the XPA.42.068 (3 and 10 mg/kg),XPA.42.089 (3 and 10 mg/kg), and also the BM-1 comparator (3 mg/kg)treated groups were significantly smaller at day 28 than the 1 mg/kgtreated groups (P value<0.05). Additionally, XPA.42.068 at 10 mg/kg andXPA.42.089 at 3 and 10 mg/kg showed significant differences compared toIgG control using Tukey's ANOVA testing (Table 8).

TABLE 8 Tumor growth inhibition in xenograft tumor model Day 28 Tukey'sMultiple Comparison t-Test Anova One TGI p value Tailed Groups % P <0.05? p-Value  3 mg/kg BM-1 vs Mouse IgG1 70.1 No 0.0089  1 mg/kgXPA.42.068 vs anti-KLH IgG2 21.3 No 0.3037  3 mg/kg XPA.42.068 vsanti-KLH IgG2 63.3 No 0.0339 10 mg/kg XPA.42.068 vs anti-KLH IgG2 99.8Yes 0.0014  1 mg/kg XPA.42.089 vs anti-KLH IgG2 51.4 No 0.0948  3 mg/kgXPA.42.089 vs anti-KLH IgG2 87.3 Yes 0.0045 10 mg/kg XPA.42.089 vsanti-KLH IgG2 93.4 Yes 0.0024

Further evaluation in the Detroit 562 xenograft model was conductedusing the antibodies XPA.42.068, XPA.42.089 and XPA.42.681. Eight tonine week old Nu/Nu mice (Charles River Laboratories) were implantedsubcutaneously with 5×10⁶ Detroit 562 cells in BD MATRIGEL™ (1:1, 200uL) per animal, into the lower left ventral abdominal region. Animalswere randomized into test groups of twelve mice each: anti-KLH humanIgG2 isotype control (3 mg/kg), XPA.42.068 (1 or 3 mg/kg dose),XPA.42.089 (1 or 3 mg/kg dose), XPA.42.681 (1 or 3 mg/kg dose), BM-1comparator (1 or 3 mg/kg dose). Dosing and tumor volume measurementswere done biweekly (FIG. 5). Animals were sacrificed the day after thelast dose (day 30), after 7 doses of antibody treatment. For allmeasurements, statistical significance was determined by one-tailedStudent's t-test.

As shown in FIG. 5 and Table 9, tumors treated with XPA.42.681,XPA.42.089, and the BM-1 comparator at 3 mg/kg showed significantdifferences in percent TGI and mean tumor volumes at day 30 comparedwith control antibody. Comparisons among these groups did not showsignificant differences using Tukey's ANOVA testing.

TABLE 9 Tumor growth inhibition in xenograft tumor model Day 30 Tukey'sMultiple Comparison t-Test Groups ANOVAs p value One-Tailed (vs.anti-KLH IgG2) TGI % p < 0.05? p-Value XPA.42.068 (1 mg/kg) 15.9 No0.3434 XPA.42.068 (3 mg/kg) 42.7 No 0.1046 XPA.42.681 (1 mg/kg) 30.4 No0.2183 XPA.42.681 (3 mg/kg) 78.7 No 0.0119 BM-1 (1 mg/kg) 20.7 No 0.3010BM-1 (3 mg/kg) 79.3 No 0.0096 XPA.42.089 (1 mg/kg) 18.5 No 0.3030XPA.42.089 (3 mg/kg) 81.8 No 0.0094

Example 12 Tumor Inhibition by TGFβ Antibodies in a Syngeneic MouseModel

The antibodies XPA.42.068 and XPA.42.089 were also evaluated for theirability to inhibit tumor growth in a syngeneic model, using 4T1 breastcancer cells, using a protocol adapted from Nam et al. (Cancer Res.68:3915-23; 2008). Balb/c female mice eight weeks of age were implantedsubcutaneously with 250,000 4T1 cells in the 4^(th) mammary fat pad onday 0. Animals were randomized into test groups of twelve mice each andadministered antibody three times per week (beginning at day −1) at asingle dose level of 10 mg/kg, with anti-KLH human IgG2 isotype control,XPA.42.068, XPA.42.089, BM-1 comparator, or mouse isotype control IgG1.Tumor volumes were measured twice weekly over the course of theexperiment and the data are shown in FIG. 6. The end of study tumorvolume data indicated that both XPA.42.068 and XPA.42.089 significantlyinhibited tumor growth as compared to the KLH control antibody.

Additionally, the animals were sacrificed on the final study day (day23) and tumors were removed to determine tumor weights. Each of theXPA.42.089, XPA.42.068 and BM-1 antibodies significantly reduced tumormass relative to the human or mouse control antibodies (FIG. 7).

Further evaluation in the 4T1 syngeneic model was conducted using theantibodies XPA.42.068, XPA.42.089 and XPA.42.681. Eight week old Balb/cmice were implanted subcutaneously with 250,000 4T1 cells in the 4^(th)mammary fat pad on day 0. Animals were randomized into test groups oftwelve mice each and administered antibody three times per week(beginning at day −1) at a single dose level of 10 mg/kg, with anti-KLHhuman IgG2 isotype control, XPA.42.068, XPA.42.089, XPA.42.681, BM-1, ormouse isotype control IgG1. Tumor volumes were measured twice weeklyover the course of the experiment and the data are shown in FIG. 8. Theend of study tumor volume data indicated that each of the antibodiesXPA.42.068, XPA.42.089, XPA.42.681 and BM-1 significantly inhibitedtumor growth as compared to the human or mouse control antibodies.

Additionally, the animals were sacrificed on the final study day (day21) and tumors were removed to determine tumor weights. Each of theXPA.42.089, XPA.42.068, XPA.42.681 and BM-1 antibodies significantlyreduced tumor mass relative to the human or mouse control antibodies(FIG. 9).

Example 13 In Vivo Effect of TGFβ Antibodies on NK Cells in Mouse TumorModel

To evaluate whether the TGFβ antibodies exhibited an immune modulatoryeffect in vivo on natural killer (NK) cells present in tumors, theisolated tumors that were removed from mice in the 4T1 syngeneic modelexperiments above were digested to generate single cell suspensions.Briefly, freshly harvested tumors were minced and digested in 2.5 mg/mLcollagenase II and 2.5 mg/mL collagenase IV in HBSS (15 minutes at 37°C.). Cells were counted and resuspended at 2e6/mL in PBS, 0.5% BSA, 0.1%NaN3 and 10 ug/mL of the 2.4G2 anti-mouse Fc blocking antibody(eBioscience, San Diego, Calif.), and incubated for 15 minutes at 4° C.After washing in PBS with 0.5% BSA, cells were stained for 30 minutes at4° C. with an anti-CD335 (anti-NKp46) antibody, conjugated forimmunofluorescent staining with flow cytometric analysis (BioLegend, SanDiego, Calif.). CD335, also known as NKp46, is a cell surface markerexclusively expressed on CD3-CD56+ NK cells, and considered to be auniversal marker for NK cells. Cells were fixed in freshly prepared 2%paraformaldehyde and analyzed on a BD FACSCanto™ system. Single colorcontrols were also prepared for compensation. As shown in FIG. 10, theXPA.42.089 antibody significantly increased expression of the NK cellmarker NKp46 (CD335) within tumors removed from mice, as compared toisotype control antibody. The BM-1, XPA.42.068 and XPA.42.681 antibodiesdid not lead to a similar increase in NKp46.

Example 14 In Vivo Effect of TGFβ Antibodies on MDSC in Mouse TumorModel

To evaluate whether the TGFβ antibodies exhibited an immune modulatoryeffect in vivo on myeloid-derived suppressor cells (MDSC) (CD11b+/Gr1+)present in tumors, the isolated tumors that were removed from mice inthe 4T1 syngeneic model experiments were prepared as described above andstained for 30 minutes at 4° C. with anti-CD11b and anti-Gr1 antibodiesconjugated for immunofluorescent staining with flow cytometric analysis(BioLegend, San Diego, Calif.). CD11b, also known as α_(M)-integrin, andthe myeloid lineage differentiation antigen Gr1, also known as Ly6G, arecell surface markers co-expressed on MDSC. Cells were fixed in freshlyprepared 2% paraformaldehyde and analyzed on a BD FACSCanto™ system.Single color controls were also prepared for compensation. As shown inFIG. 11, the XPA.42.068, XPA.42.089 and XPA.42.681 antibodiessignificantly decreased accumulation of myeloid-derived suppressor cells(MDSC, CD11b+/Gr1+) within tumors removed from mice, as compared toisotype control antibody. The BM-1 comparator antibody did not exhibit asimilar decrease in MDSC.

Example 15 In Vivo Effect of TGFβ Antibodies on Dendritic Cells in MouseTumor Model

To evaluate whether the TGFβ antibodies exhibited an immune modulatoryeffect in vivo on dendritic cells (DC) present in tumors, the isolatedtumors that were removed from mice in the 4T1 syngeneic modelexperiments were prepared as described above and stained for 30 minutesat 4° C. with anti-CD11c antibody conjugated for immunofluorescentstaining with flow cytometric analysis (BioLegend, San Diego, Calif.).CD11c, also known as α_(X) integrin, is a cell surface marker found onDC. Cells were fixed in freshly prepared 2% paraformaldehyde andanalyzed on a BD FACSCanto™ system. Single color controls were alsoprepared for compensation. As shown in FIG. 12, the XPA.42.089 antibodysignificantly decreased expression of the DC marker CD11c within tumorsremoved from mice, as compared to isotype control antibody. The BM-1,XPA.42.068 and XPA.42.681 antibodies did not exhibit a similar decreasein CD11c.

Example 16 In Vivo Effect of TGFβ Antibodies on Regulatory T Cells inMouse Tumor Model

To evaluate whether the TGFβ antibodies exhibited an immune modulatoryeffect in vivo on regulatory T cells (Treg) present in tumors, theisolated tumors that were removed from mice in the 4T1 syngeneic modelexperiments were prepared as described above and stained for 30 minutesat 4° C. with anti-CD4, anti-CD25 and anti-FOXP3 antibodies conjugatedfor immunofluorescent staining with flow cytometric analysis (BioLegend,San Diego, Calif.). CD4, also known as L3T4, as well as CD25, also knownas the low affinity IL-2Rα, and also FOXP3, also known as Forkhead boxprotein P3, are each cell surface markers found on Treg cells. Cellswere fixed in freshly prepared 2% paraformaldehyde and analyzed on a BDFACSCANTO™ system. Single color controls were also prepared forcompensation. As shown in FIG. 13, the XPA.42.068 antibody significantlydecreased accumulation of Treg cells within tumors removed from mice, ascompared to isotype control antibody. The BM-1, XPA.42.089 andXPA.42.681 antibodies did not exhibit a similar decrease in T-reg cells.

Example 17 In Vivo Effect of TGFβ Antibodies on Cytotoxic T Cells inMouse Tumor Model

To evaluate whether the TGFβ antibodies exhibited an immune modulatoryeffect in vivo on cytotoxic T lymphocyte cells (CTL) present in tumors,the isolated tumors that were removed from mice in the 4T1 syngeneicmodel experiments were prepared as described above and stained for 30minutes at 4° C. with anti-CD8 antibody conjugated for immunofluorescentstaining with flow cytometric analysis (BioLegend, San Diego, Calif.).CD8 is a cell surface marker found on CTL. Cells were fixed in freshlyprepared 2% paraformaldehyde and analyzed on a BD FACSCANTO™ system.Single color controls were also prepared for compensation. As shown inFIG. 14, the XPA.42.068 antibody significantly increased levels of CTLwithin tumors removed from mice, as compared to isotype controlantibody. The BM-1, XPA.42.089 and XPA.42.681 antibodies did not exhibita similar increase in CTL.

The results above demonstrate that the anti-TGFβ antibodies disclosedherein have the ability to decrease tumor volume size as well asmodulate immune cells that infiltrate tumors and contribute to tumorgrowth in vivo. This suggests that the anti-TGFβ antibodies describedherein will provide a therapeutic benefit in the treatment of cancer, inparticular, in cancers in which any one or more of the immune cells inthe examples above infiltrate into the tumor cells.

Example 18 Improvement in NK Cell Cytolytic Activity

A natural killer (NK) cell co-culture system was developed to mimicchronic interaction between NK cells and tumor cells in vivo, forevaluating the ability of the anti-TGFβ antibodies to improve NK cellcytolytic activity. The TGFβ producing mouse mammary carcinoma cellline, 4T1, was used. NK cells were purified from spleens of normalBalb/c mice and co-cultured with CFSE-labeled 4T1 tumor cells for 48hours in the presence of IL-2 (500 IU/ml) in 6-well plate. Anti-TGFβ andcontrol antibodies were added into the co-culture system and NK cellswere harvested 48 hours later. IFNγ production of NK cells was measuredright after the co-culture by intracellular staining. NK cells weresorted as CFSE negative cells and their cytolytic activity was analyzedby standard killing assays against the Yac-1 tumor cell line. NK cellswere co-cultured with CFSE labeled Yac-1 cells at an effector:target(E:T) ratio of 20:1 for 4 hours in 96-well round bottom plate. Propidiumiodide (PI) stain was used to mark cell death.

NK cells showed an elevated but not significant increase in IFNγproduction among antibody treated groups compared to anti-KLH treatedgroup. In the killing assays, at an effector:target ratio of 20:1, theBM-1 antibody and both the XPA.42.089 and XPA.42.681 antibodiessignificantly improved NK cell cytolytic activity (FIG. 15). Moreover,both the XPA.42.089 and XPA.42.681 antibodies increased the ability ofNK cells to kill target tumor cells to 97.8% and 96.7%, which werelevels significantly greater than the benchmark comparator (P<0.0001).This result indicates that the TGFβ neutralizing antibodies of thepresent disclosure can significantly improve NK cell cytolytic activitythat was dampened by chronic interaction with TGFβ producing tumor cellsin vitro.

Example 19 Inhibition of the Tolerogenic Function of CD8+ DendriticCells

An in vitro system based on mixed lymphocyte reaction (MLR) wasdeveloped to evaluate the ability of anti-TGFβ antibodies to inhibit thetolerogenic function of TGF-β on CD8+ dendritic cells (DC). MLR is aclassic experiment used to test DCs antigen presentation without addingexternal antigens into the system. Spleens from normal Balb/c mice werecut into small fragments and incubated in 10% RPMI and lmg/mlcollagenase type IV for 1 hour at 37° C. in a shaking incubator. Afteradding EDTA for additional 5 minutes, the solution was filtered througha nylon mesh. CD11c+ DCs were stained with biotinylated anti-CD11cantibody and positively selected using a biotin purification kit fromStemcell Technologies. CD11c+ DCs were stained with CD8 antibody. CD8+and CD8− populations were sorted on the BD FACSARIA™ cell sorter. CD8+DCs were cultured with anti-TGFβ antibodies or control antibody for 24hours and mixed into CD8− DCs at 1:10 ratio. T cells were purified fromnormal B6 spleens using a T cell negative selection kit from StemcellTechnologies and labeled with CFSE. Mixed DCs were then co-cultured withB6 T cells for 5 days in 96-well round bottom plates. The immuneinhibitory function of CD8+ DCs was evaluated by T cell proliferation.If CD8+ DCs inhibit the ability of CD8− DCs to present antigens, B6 Tcells proliferate less. If anti-TGFβ antibodies block autocrine TGFβ anddampen CD8+ DC tolerogenic function, B6 T cells proliferate more.

As shown in FIG. 16, little change was observed in T cell proliferationfor the BM-1 treated group compared to the anti-KLH control group. Incontrast, both the XPA.42.089 and XPA.42.681 antibodies significantlyincreased T cell proliferation as compared to the control anti-KLHtreated group, and the effect of XPA.42.681 on T cell proliferation wassignificant compared to the benchmark antibody BM-1. These data showthat by blocking TGF-β, the tolerogenic effect of CD8+ DCs onimmunogenic DC can be reduced, which may provide enhanced antigenpresentation by immunogenic DCs.

Example 20 Enhancement of CTL Function

An in vitro system was developed to mimic the activation of CTLs undertumor conditions and determine whether CTL function could be enhanced bythe anti-TGFβ antibodies. CTL activation was evaluated by CD25expression and function by staining of perforin and granzyme B (GzmB)(Massague et al. Cancer Cell 8: 369-380, 2005). T cells were purifiedfrom normal Balb/c spleens by T cell negative selection (StemcellTechnologies). MACs beads were coated with anti-CD3 and anti-CD28antibodies with T cell activation/expansion kit by Miltenyi Biotec(Auburn, Calif.). T cells and anti-CD3 and anti-CD28 coated-MACs beadswere co-cultured at a 1:1 ratio at a concentration of 2×10e6/mL in96-well round bottom plate in the presence of 20 ul of 4T1 culturesupernatant for 48 hours, with anti-TGFβ antibodies and controlantibody. CTL activation was evaluated with CD25 expression on the cellsurface and function was evaluated with the expression of GzmB (FIG.17A) and perforin (FIG. 17B), determined by intracellular staining usinga FoxP3 staining protocol.

Consistent with published data, changes in CD25 expression were notobserved between antibody treated groups and the control group. BM-1treated CTLs showed minimal changes in both GzmB and perforinexpression. However, both the XPA.42.089 and XPA.42.681 antibodiessignificantly increased GzmB expression in CTLs as compared to controlanti-KLH treatment (P<0.001). Moreover, the increase in GzmB expressionin XPA.42.089 treated CTLs was significantly greater than the comparatorBM-1 (P<0.05). For perforin expression, both XPA.42.089 and XPA.42.681treated CTLs produced significantly more perforin compared to controlanti-KLH treated CTLs (P<0.05). In addition, XPA.42.681 improvedperforin expression in CTLs significantly more than the BM-1 comparator(P<0.05). Thus, both XPA.42.089 and XPA.42.681 can restore theexpression of both GzmB and perforin suppressed by TGF-β secreted fromtumor cells in this in vitro culture system, and therefore provides amechanism to boost CTL function by neutralizing TGFβ produced by tumorcells.

Example 21 Inhibition of MDSC and Treg Function

An in vitro co-culture system is developed to evaluate the effect ofmyeloid-derived suppressor cells (MDSCs) on the expansion and functionof regulatory T cells (Tregs), and the ability of anti-TGFβ antibodiesof the present disclosure to inhibit MDSC and Treg activity.

MDSCs are known to suppress the immune response against tumors andpromote tumor invasion and metastasis, as well as promote the expansionand function of Tregs, which are known to down-regulate immuneresponses. Female BALB/c mice are inoculated in the abdominal mammarygland with 7 ×10³ 4T1 tumor cells in 50 ul 1× PBS. Spleens are harvestedon day 21 and MDSCs purified via biotin labeled CD11b antibody andbiotin positive selection kit (Stemcell Technologies). At the same time,normal BALB/c spleens are harvested and T cells are purified via a Tcell negative selection kit (Stemcell Technologies). The cells arestained with anti-CD4-FITC and anti-CD25-PE. Double positive cells aresorted with a FACSARIA™ cell sorter (BD Bioscience). The Treg populationis CFSE labeled and co-cultured with MDSCs from 4T1 tumor injected miceat a 1:1 ratio for 5 days in the presence of anti-TGFβ or controlantibodies. The expansion of Tregs is measured by CFSE divisions. Toevaluate the effect of MDSCs on Treg function, MDSCs harvested from 4T1injected BalB/c mice are CFSE-labeled and co-cultured with Tregs for 5days in the presence of anti-TGFβ or control antibodies. Tregs aresorted as a CFSE negative population from the co-culture. T cells fromnormal BALB/c mice are CFSE-labeled and plated at 2×10⁶ cells/ml withanti-CD3 and anti-CD28 beads. Sorted Tregs are added into the culturesystem. The inhibitory function of Tregs is measured by analyzing thenumber of CFSE divisions of the T cells.

Example 22 Inhibition of Fibrosis by TGFβ Antibodies in a Mouse Model

Antibodies of the present disclosure are also evaluated for theirability to inhibit fibrosis (e.g., lung fibrosis, kidney fibrosis) inanimal models of fibrosis.

Kidney Fibrosis

A kidney fibrosis model was used to evaluate the anti-TGFβ antibodies(Ling et al., J. Am. Soc. Nephrol. 14:377-388; 2003). Cyclosporine A(CsA, 30 mg/kg) or olive oil as vehicle control was injectedsubcutaneously once daily for 4 weeks into 6-7 week old male ICR mice ona low-salt diet (LSD, 50-100 ppm NaCl) to initiate kidney fibroticdisease. Control mice were maintained on a normal diet and did notreceive CsA. Anti-TGFβ antibody XPA.42.089 or IgG control antibody wasdosed intraperitoneally (2.5 mg/kg, TIW) beginning one day prior tocommencing CsA treatment. Animals were euthanized, and serum, urine andkidneys were collected for evaluation of histology and kidney functionendpoints.

Histopathologic examination was performed by staining formalin-fixed andparaffin-embedded kidney sections (5-μm) with hematoxylin-eosin (H&E)and Masson trichrome, using standard techniques. Assessment ofCsA-induced histopathologic changes may include commonly acceptedsemiquantitative scoring (Ling et al., Am. J. Physiol. 277:F383-F390,1999) of coded sections and assessment on the basis of any or all oftubular damage, interstitial infiltrates, thickening of arterioles,tubulointerstitial expansion, and fibrosis, including for examplescoring by counting the percentage of the diseased area per kidneysection. Sagittal kidney sections from normal control, CsA-injected andXPA.42.089 antibody treated mice were stained with Masson's trichromestain. Development of fibrosis induced by CsA was observed in thetubulointerstitium of CsA injected mice, but not in the control animals.Additionally an increase in the luminal diameter of some tubules wasobserved in CsA treated mice. Treatment with the XPA.42.089 antibodyreduced the amount of CsA-induced fibrosis observed in thetubulointerstitium and reduced tubule diameter.

Kidney function also may be evaluated by any or all of serum creatinine,blood urea nitrogen, and urine biomarkers of kidney dysfunction.

Serum blood urea nitrogen (BUN) is an indicator of kidney dysfunction.In this study, BUN was significantly increased in mice exposed to CsA ascompared to chow-fed or LSD-fed control mice (FIG. 18). Treatment withXPA.42.089 significantly reduced serum BUN compared to the IgG controlantibody.

Albuminuria, or an increase in albumin accumulation in the urine, ischaracteristic of glomerular dysfunctional in the diseased kidney. Inthis study, urine albumin was increased nearly four-fold in CsA micerelative to chow-fed or LSD-fed control mice (FIG. 19). Treatment withXPA.42.089 resulted in a significant improvement in albuminuria ascompared to IgG control antibody treated mice.

Levels of urine type IV Collagen, which reflect the extent of ECMdeposition and fibrosis in the kidneys, was significantly increased inCsA mice relative to chow-fed or LSD-fed control mice (FIG. 20).Treatment with XPA.42.089 moderately decreased urine type IV collagencompared to IgG control antibody.

Quantitative RT-PCR was performed on kidney tissue to determineexpression of genes involved in fibrosis. Total RNA was isolated fromkidneys (cortex and medulla) using the RNeasy Kit (Qiagen, Germantown,Md.) according to the manufacturer's protocol. First-strand cDNA wassynthesized using random primers and MULTISCRIBE™ RT (AppliedBiosystems, Carlsbad, Calif.). Quantitative RT-PCR was then performed on2 μl cDNA using SYBR Green mix (Roche) on the LIGHTCYCLER 480 Real TimePCR system (Roche Applied Science, Indianapolis, Ind.). Values werenormalized to cyclophilin and calculated using the comparative CTmethod. TGF-β1 is a potent inducer of fibroblast differentiation and thedeposition of ECM proteins, including type III collagen. TGF-β1expression was nearly two-fold higher in CsA-treated animals whencompared to control mice (FIG. 21A). Treatment with XPA.42.089significantly reduced the expression levels of TGF-β1 in the kidney ascompared to IgG control antibody. A similar effect was observed forexpression of type III collagen, with a moderate elevation observed inCsA treated mice (FIG. 21B). Treatment with XPA.42.089 resulted indecreased type III collagen expression levels compared to IgG controlantibody treated mice.

Lung Fibrosis

A lung fibrosis model may be used, essentially as described by Wilson etal. (J. Exp. Med. 207:535-552; 2010). C57BL/6 mice are anesthetized andinstilled intratracheally with 0.15 U bleomycin sulfate (Calbiochem, LaJolla, Calif.) in saline, with or without antibody (e.g., n=10 pergroup, 500 ug) on days −1, 3 and 5. Animals are sacrificed on day 7 foranalysis of lung histology, lung collagen content (e.g., collagendeposition), and inflammatory infiltration. For lung histology, 5-μmsections of paraffin-embedded lung tissue are stained with Masson'sTrichrome. Lung injury, measured as bronchoalveolar lavage (BAL)collagen and collagen deposition in lung, is quantified using the Sircolassay. Inflammatory infiltration is measured in the BAL by flowcytometry.

Example 23 Treatment of TGFβ Mediated Ophthalmological Disorders

Anti-TGF-β antibodies of the present disclosure may be used for thetreatment of a number of ophthalmological (i.e., eye) diseases andconditions, including for example fibrotic diseases in the eye (e.g.,diseases associated with fibroproliferative states).

Neutralization of TGFβ1 in Retinal Pigment Epithelial Cells

Maintenance of the epithelial phenotype is critical for tissuehomeostasis. In the retina, de-differentiation of retinal pigmentepithelium (RPE) leads to retinal dysfunction and fibrosis, and TGFβcontributes to retinal de-differentiation by a number of mechanisms,some of which are dependent on activation of the SMAD2 pathway.Antibodies of the present disclosure were evaluated for their ability tocounteract activation of TGFβ responses in RPE cells, using a pSMAD2assay.

Retinal Pigment Epithelial (RPE) cells (Lonza #194987) were maintainedin Retinal Pigment Epithelial Cell Growth Media (Lonza #00195409). Cellswere detached with trypsin, the trpysin was neutralized (TrypsinNeutralization Solution, Lonza #CC-5002), and the cells were pelleted,resuspended at 1e6cells/mL and plated at 100,000-200,000 cells/well intoa 6 well dish. The following day, cells were washed and RPE Basal Media(Lonza #00195406) was added to arrest cells in G0/G1 phase. The nextday, cells were treated with 10 ng/ml TGFβ1 (Peprotech #100-21)pre-incubated for 5 minutes with or without anti-TGFβ antibodiesXPA.42.068, XPA.42.089, and XPA.42.681, the benchmark antibodies BM-1and BM-2, or a control anti-KLH-G2 antibody at 10 ug/ml. After 30minutes at 37° C., cells were lysed in cell lysis buffer (Cell SignalingTechnology, Danvers, Mass.) containing 1 mM phenylmethylsulfonylfluoride (PMSF) added fresh. After rocking 5 minutes at 4° C., cellswere scraped off and dispensed into a 96 deep well plate to lyse on icefor 20 minutes. Lysates were spun down at 3K for 5 minutes at 4° C.Lysates were diluted and run according to manufacturers recommendationsfor phoshpo-SMAD2 (Cell Signaling #7348) and total SMAD2 (Cell Signaling#7244) detection.

As shown in FIG. 22, TGFβ1 treatment causes a robust increase in pSMAD2in RPE cells, which was significantly neutralized by each of theantibodies XPA.42.089, XPA.42.068 and XPA.42.681, and the benchmarkantibodies. These data suggest that XPA.42.089, 068 and 681 cancounteract TGFβ mediated signaling in RPE cells and indicates theantibodies may be useful for treatment of retinal dysfunctions.

Proliferative Vitreoretinopathy

Proliferative vitreoretinopathy (PVR) is the most common cause offailure in retinal detachment surgery. PVR is characterized by formationof fibrovascular membranes within the vitreous cavity above and beneaththe retina, causing subsequent retinal detachment. Various factorscontribute to the progression of PVR, and TGFβ is believed to play apivotal role. TGFβ is abundant in the vitreous of PVR patients, andcharacteristic functions of TGFβ, such as the induction of epithelial tomesenchymal transition (EMT), stimulation of extracellular matrixproduction, contraction of cellular membrane, and induction ofinflammation, are all negative factors in the progression of PVR.

To evaluate the effect of antibodies of the present disclosure,experimental PVR is induced in a rabbit model (Oshima et al., 2002, GeneTher. 9:1214-1220; Fastenberg et al., Gene Ther. 2002, 9:1214-1220).Adult pigmented rabbits are anesthetized with an intramuscular injectionof isoflurane or ketamine and xylazine. The pupils are dilated with onedrop of 10% phenylephrine hydrochloride, 1% tropicamide, and 1% atropinesulfate. One eye of each rabbit is injected with 5.0×10e5 rabbitconjunctival fibroblast cells in 0.1 ml BSS solution in the vitreouscavity through the pars plana. Pars plana vitrectomy will induce the PVRmodel. Immediately thereafter, a single intravitreal injection of BSS,anti-TGFβ antibody (e.g., XPA.42.089, XPA.42.681, 5 mg) or controlantibody (e.g., anti-KLH-G2) is administered to groups of 10 animals,and optionally repeated weekly. All injected eyes areophthalmoscopically examined on days 1, 3, 5, 7, 10, 14 and 28, with PVRclassified into six stages using the clinical criteria described byFastenberg et al., Am. J. Ophthalmol. 93:565-572, 1982).

Alkali Burn to the Cornea

Ocular trauma in the form of an alkali burn to the cornea is a seriousclinical problem and may cause severe and permanent visual impairment.Activation of corneal cells, i.e., keratocytes and epithelial cells, andinflux of inflammatory cells such as monocytes/macrophages, are involvedin the pathogenesis of injury after alkali tissue damage in the corneaand can lead to persistent epithelial defects. Moreover, breakdown ofthe basement membrane by matrix metalloproteinases (MMPs, gelatinases)secreted by these cells contributes to the pathogenic ulceration andperforation of the stroma. Conjunctivalization of the corneal surface onthe loss of limbal stem cells together with opacification andneovascularization of the corneal stroma all impair the patients' visionin the later healing phases. A number of growth factors and cytokines,including TGF-β, are believed to be involved in the tissue destructionand late scarring that occur in the cornea after alkali burn.

To evaluate the effect of antibodies of the present disclosure, a mousealkali burn model is used (Saika et al., Am. J. Pathol. 2005,166:1405-18). Briefly, three μl of 1 N sodium hydroxide solution isapplied to the right eye of adult C57BL/6 mice (n=72) to produce anocular surface alkali burn under both general and topical anesthesia.Anti-TGF-β antibodies (e.g., XPA.42.089, XPA.42.681) or control antibody(e.g., anti-KLH-G2) are administered (n=24/group) at 2 hours and days 5,10, and 15 after the alkali exposure. Fluorescein staining of the corneais used to visualize surface defects (e.g., injured epithelium). Aftercorneal fluroescein examination, the eye globe is enucleated 2 hoursafter labeling with bromodeoxyuridine and processed for histologicalexamination in either paraffin or cryosections at days 3, 5, 10, and 20.

Lens Fibrosis

Following injury, lens epithelial cells undergo EMT, which contributesto the formation of fibrotic tissue in the injured lens. A similarphenomenon is observed in the human lens capsule following cataractextraction and implantation of an artificial intraocular lens. Such anEMT-related fibrotic reaction is clinically unfavorable since it maycause opacification and contraction of the remaining anterior lenscapsule, as well as opacification in the posterior capsule. Eye aqueoushumor contains abundant TGF-β and a role has been suggested for TGF-β ininjury-related EMT in lens epithelial cells.

To evaluate the effect of antibodies of the present disclosure,experimental corneal fibrosis is induced in a mouse model (Saika, etal., Am J Pathol. 2004, 164:651-663). Adult mice (4 to 6 weeks old) areanesthetized with an intraperitoneal injection of pentobarbital sodium(70 mg/kg). A small incision is made in the central anterior capsulewith the blade part of a 26-gauge hypodermic needle through a cornealincision in the right eye after topical application of mydriatics andoxybuprocaine eyedrop as anesthetic. Immediately thereafter, anti-TGFβantibodies (e.g., XPA.42.089, XPA.42.681) or control antibody (e.g.,anti-KLH-G2) (n=24/group) are administered to the eyes twice weekly forthe duration of the study. The left eye serves as an uninjured control.The depth of injury is ˜300 μm, or approximately one-fourth of thelength of the blade part of the needle, which leads to the formation offibrotic tissue around the capsular break. After instillation ofofloxacin ointment, the animals are allowed to heal for 6 hours to 8weeks. Proliferating cells are labeled by an intraperitoneal injectionof bromodeoxyuridine, followed by sacrifice of the animals 2 hours laterand enucleation of each eye for analysis.

Postoperative Glaucoma Surgery

The major determinant of the long-term outcome of glaucoma surgery isthe wound-healing response. Excessive postoperative scarring at thelevel of the conjunctiva and sclerostomysites is associated with poorpostoperative pressure control. Use of the antiproliferative agents5-fluourouracil (5-FU) and mitomycin C (MMC) in such surgery can alsocause widespread cell death and apoptosis and can result in cornealerosions and cystic avascular blebs.

To evaluate the effect of antibodies of the present disclosure on theseconditions associated with glaucoma surgery, a rabbit model is used(Mead et al., Invest. Ophthalmol. Vis. Sci. 2003, 44:3394-3401).Glaucoma filtration surgery is performed on the left eyes of New ZealandWhite rabbits (12 and 14 weeks old) under general anesthesia (ketamineand xylazine). A partial-thickness 8-0 silk corneal traction suture isplaced at 12 o'clock, to gain exposure to the superior conjunctiva. Afornix-based conjunctival flap is raised, and blunt dissection of thesubconjunctival space is performed to a distance of 15 mm behind thelimbus. An MVR blade is used to fashion a partial thickness scleraltunnel, starting 4 mm behind the limbus and continuing until the bladeis just visible in the anterior cornea stroma. A 22-gauge/25-mmintravenous cannula is then passed through the scleral tunnel until thecannula needle is visible in the clear cornea. The cannula needle entersthe anterior chamber, the cannula is advanced to the midpupillary area,and the needle is withdrawn. Finally, the cannula is trimmed and beveledat its scleral end so that it protrudes 1 mm from the insertion point,and a 10-0 nylon suture is used to fix the tube to the scleral surface.The conjunctival incision is closed with two interrupted sutures and acentral mattress-type 10-0 nylon suture on a needle to give awater-tight closure. One drop of atropine sulfate 1% and betamethasonesodium phosphate 0.1%, neomycin sulphate 0.5% ointment is instilled atthe end of surgery. Animals are then randomly allocated to receive apostoperative course of subconjunctival injections (100 μL) of anti-TGFβantibody (e.g., XPA.42.089, XPA.42.681) or control antibody (e.g.,anti-KLH-G2) (e.g., 5 mg/mL; 16/group). The subconjunctival injectionsare given on days 2, 3, 4, 7, 9, 11, and 14 after surgery (day 0) undertopical anesthesia (proxymetacaine hydrochloride 0.5% eye drops, 1 dropper eye), using a 30-gauge needle. Antibody is injected 5 mm behind thelimbus at the nasal margin of the superior rectus muscle. 5-FU isadministered 180° from the site of surgery.

Measurement of intraocular pressure in both eyes is made with a handheldtonometer after topical instillation of 0.5% proxymetacaine HCl eyedrops. The conjunctival appearance and the drainage area are observed.All animals are examined by a masked observer at set times aftersurgery. Assessment of both eyes (contralateral untreated eye used ascontrol) is made daily from days 0 to 4 and thereafter at regularperiods, at least twice weekly. Bleb characteristics, including length,width, and height, are measured with calipers, and intraocular pressureis recorded. The drainage bleb vascularity characteristics are graded bydividing the conjunctival areas into quadrants and scoring theappearance (0, avascular; +1, normal vascularity; +2, hyperemic; and +3,very hyperemic). Slit lamp examination is performed to identify bothanterior chamber activity (0, quiet; 1, cells; 2, fibrin; and 3,hypopyon) and anterior chamber depth, which is recorded as deep (+2),shallow (+1), or flat (0). An assessment of the duration of cornealepitheliopathy is made after topical installation of lignocainefluorescein into the left eye and is graded according to the area of thecornea affected (0, nil; 1, <5%; 2, <50%; 3, <75%; 4, <90%; 5, up to100%). Bleb survival is taken as the primary efficacy end point. Blebfailure is defined as the appearance of a flat, vascularized, andscarred bleb in the presence of a deep anterior chamber. Bleb area andheight, anterior chamber depth and activity, and conjunctivalvascularity per quadrant are all analyzed. Tissues are also processedfor histological examination (e.g., subconjunctival collagen deposition)from some animals.

It is expected that anti-TGFβ antibodies disclosed herein inhibit TGFβactivity during fibrotic incidences in the eye thereby decreasingfibrotic deposition and improving symptoms associated with fibrosis ofthe eye.

Numerous modifications and variations in the disclosure as set forth inthe above illustrative examples are expected to occur to those skilledin the art. Consequently only such limitations as appear in the appendedclaims should be placed on the disclosure.

What is claimed:
 1. An isolated nucleic acid molecule comprising anucleotide sequence encoding an antibody that binds transforming growthfactor beta (TGFβ)1, TGFβ2 and TGFβ3 comprising: (a) a heavy chain CDR1amino acid sequence set forth in SEQ ID NO: 19; (b) a heavy chain CDR2amino acid sequence set forth in SEQ ID NO: 20; (c) a heavy chain CDR3amino acid sequence set forth in SEQ ID NO: 21; (d) a light chain CDR1amino acid sequence set forth in SEQ ID NO: 22; (e) a light chain CDR2amino acid sequence set forth in SEQ ID NO: 23; and (f) a light chainCDR3 amino acid sequence set forth in SEQ ID NO:
 24. 2. The nucleic acidof claim 1, wherein one or more nucleotide sequences encoding heavychain framework amino acids have been replaced with corresponding aminoacid(s) from another human antibody amino acid sequence.
 3. An isolatednucleic acid molecule comprising a nucleotide sequence that encodes anantibody that binds transforming growth factor beta (TGFβ)1, TGFβ2 andTGFβ3 comprising a light chain variable region and a heavy chainvariable region, wherein (a) the light chain variable region comprisesat least a CDR1 amino acid sequence set out in SEQ ID NO: 22, a CDR2amino acid sequence set out in SEQ ID NO: 23, and a CDR3 amino acidsequence set out in SEQ ID NO: 24; and wherein (b) the heavy chainvariable region comprises at least a CDR1 amino acid sequence set out inSEQ ID NO: 19, a CDR2 amino acid sequence set out in SEQ ID NO: 20, anda CDR3 amino acid sequence set out in SEQ ID NO:
 21. 4. The nucleic acidof claim 1 or 3, further comprising a nucleotide sequence encoding aheavy chain constant region, wherein the heavy chain constant region isa modified or unmodified IgG, IgM, IgA, IgD, IgE, a fragment thereof, orany combinations thereof.
 5. The nucleic acid of claim 1 or 3, whereinone or more light chain framework amino acids have been replaced withcorresponding nucleotides encoding amino acid(s) from another humanantibody amino acid sequence.
 6. The nucleic acid of claim 1 or 3,further comprising a nucleotide sequence encoding a human light chainconstant region attached to the light chain variable region.
 7. Anisolated nucleic acid molecule comprising a nucleotide sequence encodingthe antibody of claim 1 or 3 wherein the antibody binds to TGFβ1, TGFβ2and TGFβ3 with an affinity between a Kd of 10⁻⁶ to 10⁻¹² M.
 8. Anisolated nucleic acid molecule comprising a nucleotide sequence encodingthe antibody of claim 1 or 3, wherein the antibody binds to TGFβ1 andTGFβ2 with greater affinity than to TGFβ3.
 9. An isolated nucleic acidmolecule comprising a nucleotide sequence encoding the antibody of claim1 or 3, wherein the antibody neutralizes activity of TGFβ1 and TGFβ2 toa greater extent than it does TGFβ3.
 10. The nucleic acid of claim 1 or3, wherein the nucleic acid is at least 95% identical to the light chainvariable region nucleic acid sequence set out in SEQ ID NO: 7 andcomprises the CDRs set out in SEQ ID NO: 22-24.
 11. The nucleic acid ofclaim 1 or 3, wherein the nucleic acid is at least 95% identical to theheavy chain variable region nucleic acid sequence set out in SEQ ID NO:5 and comprises the CDRs set out in SEQ ID NO: 19-21.
 12. An expressionvector comprising the nucleic acid molecule of claim 1 or 3 operablylinked to an expression control sequence.
 13. A host cell comprising thevector of claim
 12. 14. The host cell of claim 13, comprising a nucleicacid molecule encoding a heavy chain and a light chain variable region,wherein the heavy chain and light chain nucleic acids are expressed bydifferent nucleic acids or on the same nucleic acid.
 15. A host cellcomprising the nucleic acid molecule of claim 1 or
 3. 16. The host cellof claim 15, comprising a nucleic acid molecule encoding a heavy chainand a light chain variable region, wherein the heavy chain and lightchain nucleic acids are expressed by different nucleic acids or on thesame nucleic acid.
 17. A method of producing an antibody, comprisingculturing the host cell of claim 13 under suitable conditions to producean antibody and recovering the antibody.
 18. A method of producing anantibody, comprising culturing the host cell of claim 14 under suitableconditions to produce an antibody and recovering the antibody.
 19. Amethod of producing an antibody, comprising culturing the host cell ofclaim 15 under suitable conditions to produce an antibody and recoveringthe antibody.
 20. A method of producing an antibody, comprisingculturing the host cell of claim 16 under suitable conditions to producean antibody and recovering the antibody.